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Won the Battle Never the War
A/N I have been working on this chapter...honestly off and on since I published the fourth chapter...
Thea glared at Achilles as he stood and walked out of the tent she hadn't moved since he had brought her back and she didn't plan on it. Not until he took her back to the walls or she figured out a plan to escape for real this time. Achilles returned with a platter of food Thea curled into herself more as her stomach growled.
"Eat." Achilles commanded, no one had spoken to her like that since she was a child commanding her to do something. She was a Princess! And captured Princess or not she would not listen to him. And so she turned away from Achilles facing the tent. "As you wish Princess." Thea stiffened she had told him herself she was a Princess-escape attempt or not-and yet he wasn't dragging her to the "King" of Kings saying how they could use her.
"Aren't you going to take me to your King?" Thea questioned contempt flashing in her eyes, Achilles eyes narrowed.
"I have no King least of all that man." Achilles retorted Thea looked down then back up at Achilles.
"You're really not going to take me to that pig?" Achilles laughed and shook his head smirking at her.
"No little Princess I think I'll keep you for myself a while longer." Thea bit her lip unsure how to feel about that, she should try to escape again though as she looked around the tent now she couldn't spot Achilles knives. He had gotten smart moved the knives probably left them with his cousin with the simple explanation of he didn't want her to kill herself. "Am I really that bad Little Princess?"
"You're a Greek you came to invade my homeland. Do you really wish me to answer that?" Thea sneered glaring at him Achilles smirked unaffected by her glare.
"Come now Little Princess it could be a lot worse." Thea paused her immediate retort, it was true Achilles had not laid a hand on her and his men in fact had defended her, he had found her trying to escape and had done nothing but bring her back to his tent and knew she was a Princess. And yet here she sat as safe as possible in a camp full of soldiers unharmed other than a few bruises from Agamemnon's men.
Hector stood on the wall watching the beach he hoped Thea if she was dead-the thought pained him- had been given the proper burial she deserved. She was just a young girl foolish enough to leave the safety of the walls. Briseis was inconsolable the poor girl blamed herself and thought everyone else did as well, if she hadn't been at the Temple of Apollo Thea would have never left the walls. But it was Hector who truly blamed himself he should have returned to Sparta and gave Menelaus Helen and Paris when he found Helen on board. Paris was his brother but he loved his sister all the more, it was Paris's fault they were in this war and Thea was captured or dead.
Andromache watched her husband carefully finally deciding to pull him from his thoughts she wrapped her arms around his waist. Hector looked at her smiling faintly brushing a strand of her hair from her face.
"Let's go to bed husband." Hector nods and allows Andromache to lead them back to their bed.
Paris sat up looking out at the beach he felt responsible for Thea's capture he knew the whispers if he hadn't brought Helen to Troy none of this would be happening now. They would have their truce with Sparta and all would be safe. Which was why Paris knew he needed to devise a plan to get Thea back and quickly at that. She had to still be alive she had to.
Thea looked up as Patroclus entered the tent Achilles had left mere moment ago though Thea doubted he was far from the tent. Patroclus smiled at her setting down across from her. He may want glory from killing her Countrymen but dammit they boy was sweet and pleasant.
"Achilles told me you almost escaped." Patroclus said Thea sighed glaring at the boy only half seriously.
"Hardly didn't get past the beach." Thea replied she liked Patroclus he reminded her of her brothers and cousins, he was friendly enough.
"Are you hungry?" Patroclus asked Thea was starving in fact she had refused to eat the food Achilles had offered and was suffering for it now. As if with a mind of it's own her stomach growled loudly. Patroclus laughed replying. "I'll take that as a yes."
"Yes." Thea took Patroclus's hand allowing him to pull her up and lead her out of the tent in search of food.
Achilles watched as Patroclus lead Thea out of the tent he trusted his cousin but he didn't trust the Princess. She was a wily one someone had gotten it in her head that if she was ever captured to escape at all costs. Then they were on here home beach she could probably navigate it better than all the Greeks combined. He didn't doubt that if she did manage to escape the Greek encampment that she would be back at the walls of Troy before he could recapture her.
Thea fought a grin as Patroclus led her to a fire she could already smell the fish cooking over the flames. The men surrounding the fire looked at them but didn't say a word. Eudorus she thought his name was stood offering his seat to Patroclus and Thea elbowing the man who had been sitting next to him. Patroclus sat pulling Thea down next to them they were both handed plates laden with fish and bread. A wine skin was passed around the fire each man taking a drink. When it reached Thea she hesitated only a moment before taking a sip of the wine passing it to Patroclus. The men were talking they weren't happy about Achilles ordering them off fighting which was understandable they were soldiers it was what they did. Then again Thea felt all the better if the Myrmidons the fiercest warriors in all of Greece weren't fighting her countrymen stood a better chance.
"Here." Patroclus said handing Thea the wine skin it had made a second round around the fire Thea took a bigger sip this time before passing it to Eudorus.
"What do you think they chances are?" One of the men questioned Thea suddenly she blinked she hadn't been paying attention to the conversation.
"Of what?" Thea questioned the men around the fire chuckled Thea scowled feeling her cheeks burn.
"Of the Greeks chances of getting behind those high walls of Troy."
"As long as Prince Hector lives they don't stand a chance in hell." Thea said she stood and walked back to Achilles tent he smirked at her as she passed. She rolled her eyes going back to her place he and Patroclus may have one this fight but she would win the war and she would get back to Troy if it was the last thing she did.
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This June the trial of Ontario dairy farmer Michael Schmidt and three other farmers for obstructing a police officer will resume. The charge stems from a 2015 raid of Schmidt's Glencolton Farms where members of a food cooperative
Berglund Appealing Case to Minnesota Supreme Court
Dairy farmer Dave Berglund of Lakeview Natural Dairy in Grand Marais has filed a petition with the Minnesota Supreme Court to hear his case after an appellate court ruled that the Minnesota Department of Agriculture
Mark Baker Running for Sheriff
When heritage breed hog farmer Mark Baker was going through his four-year battle against the state of Michigan over his challenge to the Michigan Department of Natural Resources Invasive Species Order
Judge Rules MDA Has Power to Inspect Berglund Farm
Cook County, Minnesota District Court Judge Michael J. Cuzzo has ordered that Grand Marais dairy farmer Dave Berglund must comply with an October 14, 2014 administrative inspection order Judge Cuzzo issued allowing
Raid on Bakers Green Acres
Here is a letter from a supporter of Mark Baker about yesterday's raid on Baker's Green Acres and other actions the state of Michigan has taken against Mark. The Michigan Department of Agriculture obtained a warrant to search
Eldon Hooley Convicted over City Food Code Violation in Texas
Falls Church, VA—December 16, 2015—After a two-day trial, a jury in Fort Worth Municipal Court found FTCLDF member and Grandview dairy farmer Eldon Hooley guilty on one count of operating a food
Breaking News: Ontario Raw Milk Farmer Released After Overnight Jail
Durham, Ontario—October 20, 2015—West Grey Police detained raw milk farmer Michael Schmidt on October 19, 2015 and held him overnight when he reported for booking on theft charges. The Canadian government is
Turning the Tables in Minnesota
From 2010–2014, the Minnesota Department of Agriculture (MDA) did everything it could to put Gibbon dairy farmer Mike Hartmann out of business. Whether it was raiding Hartmann's farm, impounding his truck, confiscating and destroying his products, embargoing his
Judge Rules Berglund Not in Contempt for Refusing MDA Inspection
In a ruling paving the way for a possible precedent-setting court decision on food freedom in Minnesota, Cook County District Judge Michael J. Cuzzo has denied the Minnesota Department of Agriculture's (MDA) request to hold dairy farmer Dave Berglund in contempt for refusing to
Wisconsin Supreme Court Raw Milk Cases for Food Freedom
By Gayle Loiselle
Three Wisconsin farms now have petitions for review before the state's Supreme Court—all challenging the legality and constitutionality of contracts between farmers, private citizens and private food groups to acquire or provide fresh unpasteurized
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Ek Tha Raja Ek Thi Rani 15th June 2017 Written Episode Update
Ek Tha Raja Ek Thi Rani 15th June 2017 Written Episode Update on teleshowupdates.com
Vasundra goes to hide in a slum house. Rani and police reach behind but the man there doesn't recognize Vasundra. Later, Vasundra asks about the guy. The man boasts he is Shera, his son.
At home, Rani says Vasundra wants power. They must make a plan to trap Vasundra and announce the transfer of position to her tomorrow.
Shera returns home with medicines for Vasundra. Vasundra smiles as he boasts himself to be a pretty actor. They ask Vasundra if she is a murderer. Shera loses his balance. Vasundra tells them Rani planned against her, she had an affair with his husband's friend Doshi. He called them and wanted a clarification, they killed Raaj. Shera intently hears about the twist in story. Vasundra says they accused her for killing Raaj, she had no proof and had to run away instead. Vasundra smirks they were trapped in her trick. Vasundra tells Shera that he can write the story ahead, he is the one who can save her. Vasundra asks if he knows about Raaj Singh. Shera wonders how he would know such elites. Vasundra makes him meet Raaj, she takes him towards the mirror and says this Rani's husband Raaj. She join her hands in front of Shera.
The next morning, a man comes to take Shera and his father to go to palace. He informs Rani Chauhan is taking over the charge of Amirkot, it's her crowning. Vasundra says these people are innocent, they doesn't understand how filthy is her character for being a princess. She tells Shera not to let her take this seat, it's the testimony of his acting skills. She requests him for help and save people of Amirkot from Rani.
In the palace, Rani walks downstairs. Sakshi compliments Rani to look pretty. Rani takes blessings from Anandi and Chitra. The Pandit makes her wear the turban and hands her a sword. Rani says she is incomplete without Raja, she unveils Raaj's photo and places the sword in front of it. She was shocked to see Raaj come to palace front behind.
Everyone was shocked to see Raaj. Rani runs towards him and hugs him tightly. The whole family runs towards him. Chitra cries out of elation. Anandi was also in a state of disbelief. Sakshi comes to welcome him cheerfully. Doshi asks how he ran out of the fire, and if he is here whose body was there. Rani asks Raaj what had happened that day. Vasundra walks inside from behind.
Rani tells Doshi to get hold of this woman, she must not be allowed to run away. She questions how Vasundra dare return to this palace. Shera turns to push Doshi away from Vasundra. He stands by Vasundra saying no one must dare hurt her anyway. Everyone was shocked, Vasundra smirks.
Ek Tha Raja Ek Thi Rani 16th June 2017 Written Episode Update Precap : At night, Vasundra makes sure the secret is kept a secret. In the room, Rani asks Sheru not to get away from her, she is his wife. She asks him to fill her hairline with sindoor.


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The Shift: Taking Your Life from Ambition to Meaning by Dr Wayne W Dyer (Hardback, 2010)
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- DescriptionThe Shift --a companion to the movie of the same name--illustrates how and why to make the move from ambition to meaning. Such a shift eliminates people's feelings of separateness and illuminates their spiritual connectedness.
- Author BiographyAffectionately called the father of motivation by his fans, Dr. Wayne W. Dyerwas an internationally renowned author, speaker, and pioneer in the field of self-development. Over the four decades of his career, he wrote more than 40 books (21 of which became New York Times bestsellers), created numerous audio programs and videos, and appeared on thousands of television and radio shows. His books Manifest Your Destiny, Wisdom of the Ages, There s a Spiritual Solution to Every Problem, and the New York Times bestsellers 10 Secrets for Success and Inner Peace, The Power of Intention, Inspiration, Change Your Thoughts Change Your Life, Excuses Begone!, Wishes Fulfilled, and I Can See Clearly Now were all featured as National Public Television specials. Wayne held a doctorate in educational counseling from Wayne State University, had been an associate professor at St. John s University in New York, and honored a lifetime commitment to learning and finding the Higher Self. In 2015, he left his body, returning to Infinite Source to embark on his next adventure. Website: www.DrWayneDyer.com
Key Features
- Author(s)Dr Wayne W Dyer
- PublisherHay House Inc
- Date of Publication01/02/2010
- Language(s)English
- FormatHardback
- GenrePopular Psychology
Publication Data
- Place of PublicationCarlsbad
- Country of PublicationUnited States
- ImprintHay House Inc
- Weight249 g
- Width134 mm
- Height199 mm
- Spine17 mm
- Pagination144
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- Format DetailsSewn,Cloth over boards
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Hey everyone! I'm busy getting ready to September how about you?
I'm the type of person who can't just veg out and relax on the couch all day. I need to have something to do or someplace to go. I'm starting a new series focusing on different DIY (Do It Yourself) projects you can complete over the weekend.
The first project I want to focus on is a great back to school idea!
DIY Pocket Charts
As some of you may know I recently changed my classroom decor from Dots on Turquoise to bright Chevron. I was able to keep some of my cute creations. Yay!!!! I am keeping teal/turquoise in my new color pallet but in order to add the other colors I had to recreate some of my classroom favorites.
I know they make really cute Chevron library pockets at the teacher store, but my little Kinders will not be able to read the labels (picture clues are a must in my class). This year I recreated my Class Job Chart using images I found on Mycutegraphics.com Picture clues help my students understand their job function and take ownership of the classroom. So lets get started!!
Poster Board:
Envelopes (3 X6)
X-acto Knife
After you decided what type of pocket chart you need, you have to design the outside of your pockets. I used Photo Shop to easily customize my images. Once your images are complete you have two options...
- A print the image directly to the envelop saving you time.
- B print the image on paper and then cut and glue it to the envelope.
Run your poster through your local laminator. I would suggest using a slower setting so that the pockets don't lift up and to guarantee a tight seal.
Some of you may be thinking... WAITE!!!! How am I supposed to use the pockets if they are sealed by lamination? This is where the X-acto Knife comes in. You have to carefully slit open the top of the pockets so that you can use them.
Thanks for stopping by and good luck on your new DIY Saturday project.
Check out Teacher Times Two to see how other teachers are getting ready for September.


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THE LITTLE RED LIGHTHOUSE AND THE GREAT GRAY BRIDGE — a new musical by Jessica Ann Carp — is adapted from the 1942 children's book by Hildegarde H. Swift and Lynd Ward. This timeless New York City story tells the tale of the Little Red Lighthouse who felt important and useful until the Great Gray Bridge (based on the George Washington Bridge) was built. Then he felt very, very small and no longer needed. But then the Great Gray Bridge explained, "Each to his own place little brother!" urging the Lighthouse to see that everyone, no matter how small, has a purpose.
For this adaptation, Carp has woven new historical touches into the story. In added scenes, the Lighthouse's caretaker is revealed to be a veteran of World War I, while the Canoe stands up for women's rights. With charming characters, exciting music, and a simple but important message, this concert has been described as "a children's musical that's for grown-ups, too."
Music directed by Patrick Sulken and with direction and projection design by Brian Freeland, this truly shines!
Come celebrate the 75th anniversary of The Little Red Lighthouse and the Great Gray Bridge's original publication with this delightful musical take on a piece of classic children's literature.


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Meet your new trustees, editors-in-chief and executive vice president in the Fall issue of ARVONews
Read more
Journal of Vision
Noise masking of White's illusion exposes the weakness of current spatial filtering models of lightness perception
Assessment of Visual and Chromatic Functions in a Rodent Model of Retinal Degeneration
Outer Segment Thickness Predicts Visual Field Response to QLT091001 in Patients with RPE65 or LRAT Mutations
Schepens Eye Research Institute/Massachusetts Eye and EarBoston, Mass.
The University of Rochester Rochester, N.Y.
University of California, Irvine School of MedicineIrvine, Calif.
SBIR grantees Simon Barriga, PhD and Peter Soliz, PhD get a "second education"
Originally published in the Spring 2015 issue of
Shortly after submitting multiple National Eye Institute (NEI) Small Business Innovation Research (SBIR) applications and ARVO Annual Meeting abstracts, Simon Barriga, PhD, and Peter Soliz, PhD, sat down with ARVONews to talk about their company, VisionQuest Biomedical.
Barriga, chief research scientist, and Soliz, president and CEO, discuss the past, present and future of their startup.
ARVONews: How did VisionQuest Biomedical get started?
Barriga: Pete and I had been working together in the private sector for many years before VisionQuest. He had an idea for creating a system for diabetic retinopathy screening, so he left the company we were working for, submitted an SBIR application and that's what got the company started.
Soliz: I met Simon 12 years ago, when he was a brand new grad student at the University of New Mexico. There was a certain amount of serendipity, running into Simon and his professor at the time. Both were specialists in computer-aided image analysis, and it was a matter of applying what they were already doing to a specific problem I was interested in — diabetic retinopathy. This collaboration resulted in technologies that were patented with the university and that were the foundation of VisionQuest Biomedical.
ARVONews: What products does VisionQuest have coming down the pipeline?
Barriga: The furthest along is our software to detect diabetic retinopathy from images taken with a retinal camera, called the Diabetic Retinopathy Referral Analysis Computer System (DR-RACS).
The software works with cameras on the market right now. The process is that a photographer takes an image of a patient's retina at a primary care physician's office; our software then processes that image. We can triage that patient by determining if they're OK or if they need to visit an optometrist or an ophthalmologist for further care.
Another project is to develop our own lowcost, portable retinal camera. Our goal is to create a small camera that would be easy to use by a medical assistant and would cost under $3,000. Eventually, we'd like to take the camera and combine it with all the software we're developing and integrate it into one solution.
Finally, we have some other projects at different stages of development. We are developing software to detect retinopathy of prematurity. We are also testing software to detect vascular abnormalities in the retina that can be a sign of cardiovascular disease, as well as software to detect signs of malaria in the retina.
ARVONews: Do you have a timeline for getting DR-RACS on the market?
Soliz: We currently have a working prototype, and we have a clinical study that is ongoing. We are collecting data from several hundred subjects with several different cameras and retina specialists reading the images. We are going to use that data to validate our performance and form the basis of our submission to the Federal Drug Administration (FDA). The goal is to have the submission for the FDA early next year.
ARVONews: SBIR grants are extremely competitive. What do you think makes VisionQuest stand out to the reviewers?
Barriga: When we started, we were approaching the medical problems purely from an engineering point of view. We did not know all the barriers — clinical, regulatory, reimbursement — that stand between technology and commercialization.
And that was reflected in some of the early, unsuccessful grants we wrote. Now, we have a much more holistic view of what needs to be done to move a technology to the marketplace, and provide all that information — economics, medical need, commercial potential — in our SBIR proposals. The reviewers are becoming more and more knowledgeable about these commercialization issues, and they need to see we are prepared to overcome them.
ARVONews: Besides the SBIR grants, where else does VisionQuest receive its funding?
Soliz: So far, we have run almost exclusively on SBIR grants. But, we cannot expect the SBIR program to take us to the next step, which is getting through the regulatory process with the FDA. In some cases, the money required to do that can be on the order of $3 million – $5 million. That really requires finding outside investors. Fortunately, we are currently talking to a number of potential investors that are interested in helping us get to that next step.
I have found that developing an elevator pitch, maybe two minutes long, to convince an investor to make a 30-minute appointment with us has been very important. Once we have that appointment, Simon and his crew comes in to convince investors that we have a technology that will sell.
ARVONews: That must require a very different type of talk than what you would give at an ARVO Annual Meeting.
Barriga: Yes, and it was difficult to learn how to give those talks. You have to be prepared to answer questions of a different nature, things an academic would not ask. I had to learn how to describe the value of our technology through the medical problem it would solve and how much money people could pay to get our solution. It took several years of hanging around where people were giving these types of talks and listening. It has been a second education, really — after the PhD.
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text: To underscore how important it is for grown-ups to set an example, York University psychology Professor Debra Pepler suggests every Canadian adult should watch Children See, Children Do, a 90-second public-service clip from Australia that shows children variously mimicking an adult who is giving another adult the finger; being racist to a sales clerk; and walking by a mother struggling with spilled groceries and a stroller without offering to lend a hand. "It's absolutely a social problem, not a school problem – it just happens at school because that's where we aggregate children," said Pepler, who is also a senior scientist with the Hospital for Sick Children, in the Toronto Star Nov. 22. As Canada's foremost expert on bullying, she suggests the pace of modern life, the pressure of financial worries and the lack of old-fashioned family help in today's far-flung world combine to leave many of us with too little time to think of others. Read full story.


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tag:blogger.com,1999:blog-8908605797601657768.post533502574627762379..comments2013-01-11T12:07:29.411-08:00Comments on Blog This!: Talents hidden in the roughDorahttp://email@example.com:blogger.com,1999:blog-8908605797601657768.post-17198987307745614872012-04-03T06:22:17.616-07:002012-04-03T06:22:17.616-07:00Thanks so much Dora for your kindness! You are an...Thanks so much Dora for your kindness! You are an awesome artisan as well! So glad I was able to meet you and many others through HMC!SarahsFabCreationshttps://firstname.lastname@example.com:blogger.com,1999:blog-8908605797601657768.post-29778527840783651302012-03-31T11:41:15.414-07:002012-03-31T11:41:15.414-07:00Wonderful post! Each shop is so deserving of the s...Wonderful post! Each shop is so deserving of the spotlight they have been given. Such talented folks, such good people, and so glad you are all not just my team-mates but my friends as well♥♥Marsha Bourquinhttps://email@example.com:blogger.com,1999:blog-8908605797601657768.post-88718158957125593052012-03-31T11:17:52.333-07:002012-03-31T11:17:52.333-07:00I look forward to reading your blogs, Dora! I&...I look forward to reading your blogs, Dora!

I'm still in love with stunning quilt I bought from 'Crazy Jayne' and the three business card holders. She's a true seamstress when it comes to sewing!

Sarah's work is wonderful as well. Ya gotta love the dinosaurs!

I like your work as well. It's unique and my kinda style! Can't please everyone, so you gotta please yourself - know what I mean? :-))

LucindaCindy Myer & my shop: thisartofmine.etsy.comhttps://firstname.lastname@example.com:blogger.com,1999:blog-8908605797601657768.post-62272064450025175182012-03-31T11:09:05.610-07:002012-03-31T11:09:05.610-07:00The are some very awesome shops!!! ..and really wo...The are some very awesome shops!!! ..and really wonderful ladies (you included, dora!!) ♥Erin Kellyhttps://firstname.lastname@example.com:blogger.com,1999:blog-8908605797601657768.post-34473838132832593152012-03-31T09:56:34.856-07:002012-03-31T09:56:34.856-07:00Thank you so much for this awesome post! I am so ...Thank you so much for this awesome post! I am so honored! I appreciate the love, the business, but most of all...you Dora! You are an exceptional woman and I am happy to have you as a friend!jaynehttps://firstname.lastname@example.com:blogger.com,1999:blog-8908605797601657768.post-30034695316546033852012-03-31T09:31:54.757-07:002012-03-31T09:31:54.757-07:00Great post Dora and I agree these are some of my f...Great post Dora and I agree these are some of my favorites. Twiggy & Opal makes beautiful custom baby blankets and quilts among other things, and the clutch I bought from GoGreenWithRed is so well made and functional. I have not bought from SarahsFabCreations YET but I love her custom storage baskets. Congrats to all you talented ladies!Denihttps://email@example.com


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After years of fundraising and several construction delays, the Stone Family Playground at Ginn Field will have a grand opening celebrating on Sunday, April 28 from 11 a.m. –1 p.m.
After years of fundraising and several construction delays, the Stone Family Playground at Ginn Field will have a grand opening celebrating on Sunday, April 28 from 11 a.m. –1 p.m.
The party will include refreshments, music, prizes and some fun family games to officially open the playground, which was completed in October 2012.
"Sunday's celebration will be the perfect time to show Winchester and its residents what this community can accomplish when it works together towards one common goal." said BeGINN Again committee member Mary Ellen Rourke-Falvey.
Many families were frustrated by the numerous construction delays, fellow committee member Cara Zimmerman said.
"Once these families see the new Stone Family playground set next to the river and the new basketball courts, they will realize the delay was worth it," she said.
Many of the features that were beloved in the old playground have been retained in the newer version of Winchester's only public in-town playground (not connected with a school), including the ever-popular tire swing and sandbox. New features that are sure to become favorites include a metallophone and spider net.
"We are hoping that the new Stone Family playground at Ginn Field will serve as a meeting place for many young families," said Wolcott Street resident and fellow BeGINN committee member Melissa Noble. "When I first moved to town, I met some of my closest friends while pushing my daughter on the old tire swing. We hope this new playground fosters these same types of friendships"
The playground is named in honor of Laura Stone Mortimer; her mother, Ellen Stone; her 4-year-old son, Thomas "Finn" Mortimer V; and her 2-year-old daughter, Charlotte Mortimer, who were killed in 2010 by Laura Stone Mortimer's husband, Thomas Mortimer IV. Mortimer pleaded guilty to the four murders in October 2012 and is serving four consecutive life sentences.
For more info, email firstname.lastname@example.com


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text: Holly is amazing. We used Perfectly Posh for our Sept 6 2015. Even before we hired her she did a site visit at my family home to help decide whether a wedding was feasible there. She helped us avoid a venue that we likely would have regretted choosing. For design challenged couple like us she was invaluable in bringing together our Seattle themed wedding. It turned out beautiful. And day of she kept everyone on track and everything running Smoothly. Highly worth the investment. She is professional, responsive, and also someone you would want to have a beer with… which you can when you come to her office to go over all the details. Although she comes across as more of a champagne gal. Very Posh 🙂 We're so happy we went with Holly and her team. Highly recommend!


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I grew up without cousins. Well, I did have cousins. I knew I had cousins. I had even met three of them. But I didn't know them well, and I didn't even live in the same state as any of them. I was well into my 40s by the time I started getting to know … Continue reading Are You My Cousin?
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Once I decided to share the recipe, I realized I would need to figure out exactly how Bernie and I are related. Given the small-sized communities in the region where my father was born, It becomes pretty obvious that anyone with a common surname is going to be related. Bernie was very obliging . . .
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In Poland, Christmas Eve is a day first of fasting, then of feasting. The Christmas Eve feast begins at the appearance of the first star. There is no red meat served but fish, usually carp. The supper, which includes many traditional dishes and desserts, can sometimes last for over two hours. It is followed by the exchange of gifts. The next day, the Christmas Day, is often spent visiting friends and family. In Polish tradition, people combine religion and family closeness at Christmas. Although gift-giving plays a major role in the rituals, emphasis is placed more on the making of special foods and decorations.
As mentioned before on the night of Christmas Eve, so important is the appearance of the first star in remembrance of the Star of Bethlehem. On that evening, children watch the sky anxiously hoping to be the first to cry out, "The star has come!" Only after…
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A general paradigm to understand protein function is to look at properties of isolated well conserved domains, such as SH3 or PDZ domains. While common features of domain families are well understood, the role of subtle differences among members of these families is less clear. Here, molecular dynamics simulations indicate that the binding mechanism in PSD95-PDZ3 is critically regulated via interactions outside the canonical binding site, involving both the poorly conserved loop and an extra-domain helix. Using the CRIPT peptide as a prototypical ligand, our simulations suggest that a network of salt-bridges between the ligand and this loop is necessary for binding. These contacts interconvert between each other on a time scale of a few tens of nanoseconds, making them elusive to X-ray crystallography. The loop is stabilized by an extra-domain helix. The latter influences the global dynamics of the domain, considerably increasing binding affinity. We found that two key contacts between the helix and the domain, one involving the loop, provide an atomistic interpretation of the increased affinity. Our analysis indicates that both extra-domain segments and loosely conserved regions play critical roles in PDZ binding affinity and specificity.
Author Summary
Protein interactions play crucial roles in all biological processes. A common way of studying them is to focus on sub-parts of proteins, called domains, that mediate specific types of interactions. For instance, it is known that most PDZ domains mediate protein interactions by binding to the C-terminus of other proteins. Humans have more than 200 slightly different copies of these domains. At the level of the binding site, PDZ domains look quite similar. This is in apparent contradiction with their heterogeneous binding specificity. Using detailed molecular dynamics simulations in conjunction with statistical analysis, we predict that contacts outside of the canonical binding site play important roles in regulating protein interactions. Some of these contacts influence the overall dynamics of PDZ domains, providing an explanation for their allosteric effect. These interactions involve regions of the PDZ domains that are much less conserved, suggesting that they can help in differentiating selectivity in this large domain family.
Citation: Mostarda S, Gfeller D, Rao F (2012) Beyond the Binding Site: The Role of the β2 – β3 Loop and Extra-Domain Structures in PDZ Domains. PLoS Comput Biol 8(3): e1002429. https://doi.org/10.1371/journal.pcbi.1002429
Editor: Ruth Nussinov, National Cancer Institute, United States of America and Tel Aviv University, Israel, United States of America
Received: November 16, 2011; Accepted: January 30, 2012; Published: March 8, 2012
Copyright: © 2012 Mostarda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by Excellence Initiative of the German Federal and State Governments and EMBO long-term fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
PDZ domains are modular protein interaction domains specialized in binding short linear motifs at the C-terminus of their cognate protein partners [1], [2]. In human, they are found in hundreds of different proteins and are mostly involved in cell-cell adhesion and epithelial junctions [3]. PDZ domains are often classified on the basis of their preferred C-terminal ligand sequences. Early studies organized binding specificity in three canonical classes: class-I involving C-terminal motifs of the type [x–(s/t)–x–(v/i)cooh], class-II [cooh] and class-III [x–(d/e)–x–cooh], where is a hydrophobic residue and x any amino acid [2], [4]. This classification, though consistent with the highly conserved binding groove [2], does not explain the large selectivity observed both in naturally occurring C-terminal peptides and synthetic peptide library screening [5]–[8]. Systematic investigations of PDZ domain specificity revealed that more distal C-terminal peptide residues are involved in the binding process [7], [9], suggesting a role for the loop following the binding site [10]–[15]. For example, the solution structure of the second domain of the hPTP1E protein showed that this loop interacts with the sixth amino acid from the peptide C-terminus [10], while possible electrostatic contacts between the loop and peptide amino acids up to position eight were found in the Par3 PDZ3-VE-Cad domain [14], [15].
It was recently suggested that specificity beyond the canonical classes can be obtained by long-range interactions involving non-conserved structural motifs specific to the domain [16]. For instance, the extra-domain helical extension characterizing the third PDZ domain of PSD95 (also called DLG4 or SAP90) was shown to influence binding [17]. Although this helix is away from the binding groove, affinity is reduced by 21-fold upon truncation of this non-conserved structural motif. Titration calorimetry measurements indicated that the free-energy penalty is entropic in nature. It was proposed that enhanced side-chain flexibility upon helix truncation, which is subsequently quenched by peptide binding, might be the main reason for this effect. This exquisitely dynamical behavior, calling for a hidden dynamic allostery [17], [18], pinpointed the importance of conformational entropy upon binding mediated by structural elements not directly evident from structural inspection alone [19], [20].
Here, we investigate the set of interactions beyond the binding site influencing peptide binding in the PSD95-PDZ3:CRIPT complex. Molecular dynamics (MD) simulations indicate that residues upstream of the 4th C-terminal amino acid are crucial for binding. Specifically, lysines residues at position −4 and −7 in the CRIPT peptide are observed to dynamically interact with the loop. Shorter peptides spontaneously unbind from the domain, indicating that canonical interactions within the binding site are not sufficient for binding. Further simulations of the DLG1-PDZ2:E6 complex suggest a wide spread presence of such peptide-loop interactions in the PDZ family. Finally, we find that the extra-domain helix of PSD95-PDZ3 helps stabilizing the loop via ionic interactions. Our results provide direct evidence of the role played by peptide amino acids away from the C-terminus and the interplay with previously unrecognized PDZ structural motifs.
Protein-Ligand contacts beyond the binding site
Seminal X-ray crystallography experiments on the third PDZ domain of PSD95 in complex with the CRIPT C-terminal peptide indicated that peptide binding is realized through the last four residues (peptide positions 0 to −3), while the rest of the peptide is mostly disordered [1] (the system was crystallized with a 9-mer peptide, see below). This observation suggested a minor role of residues upstream of the last four ones for binding. To test this hypothesis, four MD simulation runs were carried out using a 5-mer peptide from CRIPT (-KQTSV-COOH, CRIPT5), a natural class-I binder of PSD95-PDZ3 (see Methods) [1], [17]. Unexpectedly, all the four runs showed spontaneous unbinding within the first 110 ns (see blue and light-blue lines of Fig. 1 for two unbinding trajectories and Table S1 for specific unbinding times and simulation lengths). Weak affinity was a somewhat surprising result, suggesting that canonical class-I interactions alone are not sufficient for binding. Interestingly, one of the runs showed rebinding from a partially unbound state. This event was mediated by the interaction of on the peptide with on the loop following the binding site as shown in Fig. S1. The same peptide with a charged N-terminus (CRIPT5*), which can reinforce this type of electrostatic interactions, remained anchored to the binding site for the total simulation time [21]. However, the peptide canonical contacts were only partially formed (see Fig. S2).
Blue and light-blue curves show two sample unbinding trajectories of the 5-mer peptide CRIPT5. The red curve shows the time series for the longer 9 residues peptide CRIPT9.
These observations suggested that interactions beyond the canonical class-I motif are needed to achieve stable binding in native conditions (i.e. without an artificially charged N-terminal peptide), possibly with a major role of the loop. To elucidate this point, four simulations with a longer 9-mer CRIPT peptide (-TKNYKQTSV-COOH, CRIPT9) were performed for a total of roughly 700 ns. The peptide remained bound to the original X-ray configuration in all runs (see red curve in Fig. 1 for a typical RMSD time trace). Strikingly, the four extra amino acids strongly influenced binding. The two lysines at peptide positions −4 and −7 transiently formed specific salt-bridges with two negatively charged loop residues, and . These contacts are dynamic, interconverting between each other on the ns time scale. On the other hand, their cumulative contribution is large: the loop and the ligand are in contact via salt-bridges for 44% of the time. These results indicate an unexpected and biologically relevant role of this loop, going beyond class-I interactions.
Structural cluster analysis provides a quantitative classification of the non-canonical interactions (see Methods for details). In Fig. 2, structural ensembles characterizing the three most populated peptide-loop configurations are shown. We used a simplified code to classify the peptide-loop interactions. At the first, second and third position there is a "1" if interactions −7∶331, −7∶332 or −4∶331 are formed, respectively; "0" otherwise (these three contacts are the statistically more relevant ones). For example, "110" indicates that peptide is in contact with both and , as shown in Fig. 2e–f. The most observed configurations are "110", "001" and "100", having a relative population of 13%, 10% and 8%, respectively (see Fig. 2 for their structural characterization; the cumulative 44% is obtained by summing up the remaining peptide-loop interacting conformations).
Specific ionic interactions between the peptide and the loop are displayed in panels (b), (d) and (f). (a–b) interacting with of the loop. (c–d) interacting with . (e–f) interacting with both and . The strings "001", "100" and "110" encode the interaction patterns (see Results).
This scenario is represented in Fig. 3 by the transition network of the different peptide-loop configurations (see Methods). Multiple pathways are present, where a quite unspecific network of conformational changes stabilizes peptide-loop interactions on a time scale which is faster than unbinding (for example, was measured for another member of the PDZ family [22]). Interestingly, the presence of peptide-loop interactions strongly influence the propensity to form canonical class-I contacts (see Fig. S2).
The loop, the N-terminal part of the ligand and a schematic representation of the interactions between them are shown in yellow, light-blue and red, respectively. Node surface and edge thickness are proportional to the population of the configuration and the total transition probability, respectively. For each node, the three-letter string of the most populated configuration is indicated. Other minor configurations are shown in parenthesis, when present.
The dynamic nature of the interactions explains why peptide-loop contacts were difficult to detect by previous structural experimental investigations [1], [13]. For instance, both the original PDZ3 X-ray structure reported by McKinnon and collaborators [1] as well as further attempts by other groups (e.g. PDB-ID:1TP3) indicated that only a four residue C-terminal stretch (positions 0 to −3) is directly involved in binding. However, this observation is not supported by in vitro evolution and mutagenesis studies [7], [9], [15]. Along the same line, titration calorimetry experiments provided evidence for the role of peptide positions beyond −3 for both affinity and specificity [23], while water-mediated interactions were found when bound to the oncogenic E6 peptide [13]. Our observations reconcile these two views, providing a unifying picture for peptide binding to PSD95-PDZ3. While a dominant configuration characterizing the interactions between the peptide and the loop is absent, the cumulative effect of these interactions is necessary for binding. This effect is mostly dynamical, indicating that structure alone does not suffice to understand function in this case.
Microscopic origin for the binding entropic penalty in the truncated form of PDZ3
PSD95-PDZ3 is characterized by an extra-domain helix at the C-terminus [1], [17]. Structural analysis of our MD data showed that the helix directly interacts with the loop as well as with a region distant from the binding site, via two salt-bridges (red dashed lines in Fig. 4a). The first one involves at the end of the helix and a negatively charged amino acid on the loop, . The second ionic interaction is between helix and , which is located in a region of the domain without specific secondary structure. This region (blue in Fig. 4a), in turn, is in spatial contact with the carboxylate binding loop. No specific helix-peptide interactions were found, only unstable hydrophobic contacts.
(a) Two strong ionic interactions are formed between the helix and the PDZ domain (red dashed lines). Magenta and blue structures correspond to residues 318–323 (the carboxylate binding loop) and 342–357, respectively. (b) Backbone RMSF differences between the WT and the helix truncated form in the bound state. The RMSF time window is of 200 ns. (c) Backbone RMSF differences in the apo state. Red, light grey, grey and black lines correspond to RMSF calculated on time windows of 200, 10, 6 and 3 ns, respectively. The lack of the helix enhances the flexibility of both the loop and the carboxylate binding loop. The latter (structure in magenta) couples with the region containing the helix interacting residue 355 (structure in blue). The enhanced backbone fluctuations do not appear at time scales faster than 10 ns (grey and black lines), the typical time range accessible by NMR spin relaxation techniques.
Recent experiments indicated that the extra-domain helix strongly influences the dynamics of the domain [17]. Binding affinity to the 9-mer CRIPT peptide was shown to decrease by 21-folds upon helix truncation through a purely entropic effect. The truncated form of PDS95-PDZ3 is defined by residues 306–395, and referred to as throughout the text [17]. To provide atomistic insights into this mechanism, MD simulations of bound to CRIPT5 and CRIPT9 were performed (see Table. S1). The short 5-mer peptide unbound very quickly () from the domain in all the four simulation runs, while CRIPT9 remained in the binding site. As observed for the WT, binding is stabilized by a network of dynamic salt-bridges between the ligand and the loop (see Fig. S2).
Analysis of the backbone root-mean-square-fluctuations (RMSF) in the WT showed that the flexibility of the bound form is not affected by helix truncation (Fig. 4b). However, it affects the unliganded (apo) form, enhancing the overall domain backbone flexibility (Fig. 4c). The enhanced flexibility is mainly localized in three regions: the carboxylate binding loop (residues 318–323), the loop (residues 330–336) and residues 341–356. The latter corresponds to the region where the helix is forming the salt-bridges with . In our simulations for the WT, this interaction is present 49% and 41% of the time in the apo and bound forms, respectively. Given the spatial vicinity between this region (i.e., 341–356) and the carboxylate binding loop, we assume that the peaks relative to these two regions are coupled, arising from the missing interaction with the helix. Similarly, the enhanced flexibility of the loop is induced by the missing interaction with the extra-domain helix through the salt-bridge between and . This interaction is very stable in both the apo and peptide-bound states, being formed 83% and 82% of the time, respectively.
These observations have important consequences for the interpretation of the entropic penalty upon binding to . Given that the flexibility of the bound form is unaffected by helix truncation, while it is much larger in the apo form, peptide binding to requires the quenching of the three regions reported in Fig. 4c and described above. Hence, our results suggest that the quenching of both the carboxylate and loops is responsible for the entropic penalty. Nevertheless, we cannot fully exclude other effects like a contribution from side chain dynamics, since decoupling entropy into local terms is a controversial and unsolved problem [24], [25].
The important role of backbone dynamics is in contrast with recent NMR relaxation experiments which found a negligible contribution of the backbone compared to side chains flexibility [17]. We suggest that this apparent contrast can be solved by looking at the time scales of the fluctuations reported in Fig. 4c. RMSF differences peaks vanish when the time windows used for the calculations are similar to the ones relevant for NMR measurements, i.e. of the order of 10 ns or less (grey and black lines in Fig. 4c). Our data indicates that the relevant backbone fluctuations are on the 100 ns time scale. Such dynamics is, on the one hand too slow to be detected by NMR spin-relaxation techniques (i.e. ) [19], [25], [26] and, on the other hand, too fast to show up as a separate subpopulation in NMR relaxation-dispersion experiments (i.e. ).
Hydrophobic stabilization of the extra-domain helical extension and the role of
Stabilization of the extra-domain helix is further mediated by a hydrophobic patch, formed by and on the PDZ domain, and and on the helix, as shown in Fig. 5a. Analysis of all human PDZ domains (see Methods) revealed that, while position 337 largely consists (i.e. 86%) of aliphatic or aromatic residues, position 328 is less conserved, with a large portion of aliphatic amino acids (see Fig. S3). Free-energy calculations between this helix and the PDZ domain performed with FoldX [27] (see Methods) predict that V328A and V328I mutants in the apo-form have a of 1.35 and −0.79 kcal/mol, respectively. Hence, mutation to ALA destabilizes the domain. MD simulations of both mutants are consistent with this scenario. Given the direct interaction between the extra-domain helix and the loop (Fig. 5b), it is found that bulkier aliphatics make this loop more rigid, avoiding the peptide induced quenching upon binding described in the previous section. Reversely, loop flexibility of the V328A mutant increases, approaching the one obtained in absence of the extra-domain helix (, blue line). These results suggest a correlation between bulkier aliphatics at position 328 and the presence of an extra-domain helix.
(a) Four amino acids form the patch. Pairwise distances are on the order of 5 Å. (b) Backbone RMSF of the loop upon different mutations. WT is shown in orange. V328I and V328A mutants are displayed in red and light-blue, respectively. The helix truncated form is shown in blue. (c) Helical propensities for the extra-domain C-terminal region of all human PDZ domains. Average helical propensity is displayed for domains with ILE/LEU/VAL at position 328 (red curve, average over 65 domains) and with ALA at position 328 (grey curve, 31 domains). The PSD95-PDZ3 extra-domain helix region is indicated as a grey box.
To further investigate this hypothesis, we used PSIPRED [28] to compute the helical propensity of C-terminal segments in all 258 human PDZ domains (see Methods). A larger helical propensity is found for domains with ILE, LEU or VAL at position 328, compared to the ones with ALA (see Fig. 5c). For instance, around 10 residues downstream of the C-terminus, an helical propensity twice as large is found (P-value of 0.02, see Methods). These results correlate very well with our previous findings, indicating that large aliphatic side chains at position 328 can serve as anchors for extra-domain segments, stabilizing the loop. Consequently, domains with an alanine at position 328 are less likely to have an extra-domain helix and we expect that in those cases the loop would be structured differently with respect to PSD95-PDZ3. This is in agreement, for example, with both PDZ1 and PDZ2 of PSD95. These domains are known to lack the C-terminal extra helix, possess an alanine at position 328 and have a different composition of the loop (see next section).
Generalization to other PDZ domains
The PSD95-PDZ3 loop (together with V328) and the extra-domain alpha-helix are remarkably well conserved in orthologs up to fly (and even partially conserved in worm), as well as in human paralogs such as SAP97 (DLG1), PSD93 (DLG2) or SAP102 (DLG3), see Fig. S4. In particular, the three charged residues involved in peptide binding and helix contact are conserved in almost all cases, providing indirect evidence that the same loop-mediated protein/ligand recognition is taking place in distant organisms. This is not the case when looking at the entire PDZ family, where the loop is highly heterogeneous both in length and amino acid composition. For instance, the loop of the PSD95-PDZ2 is more rigid, making self-interactions with the main domain body in a region close to the hydrophobic patch mentioned earlier [29]. Despite these differences, there are studies suggesting a role of the loop in binding to PDZ2. Large chemical-shifts were measured in the loop region upon binding, substantially contributing to affinity [29]. Finally, X-ray crystallography of PDZ2 from the human paralog DLG1 in complex with the oncogenic E6 peptide pointed out to an asparagine on the loop () interacting with the ligand backbone at position (using our notation) [13].
To provide a dynamical picture of the process, we performed additional simulations of the DLG1-PDZ2:E6 complex (see Methods). Our calculations reiterate the importance of for binding to PDZ2. It is found that the E6 peptide is in contact with the loop through mainly three interactions, :, : and :, for a total of 69% of time. An example structure is shown in Fig. 6. These contacts interconvert on a ns time scale. Together with the results obtained for PDZ3, these observations suggest that the loop is actively involved in binding specificity: a property that would need to be consistently explored throughout the entire PDZ family.
The peptide interacts with the binding loop via two hydrogen bonds, involving the backbone oxygen of with the side chain of and the side chain of with the backbone nitrogen of . These two interactions occur simultaneously for 22% of the simulation time.
In PDZ binding, the relatively limited information about peptide amino acids more distant from the C-terminus prevented a clear structural understanding of the effect and importance of these upstream side chains. Our work aims to fill this gap by providing calculations with both a canonical 5-mer CRIPT peptide as well as a longer 9-mer peptide in complex with PSD95-PDZ3. Three main results emerge from our work.
First, we observe in our simulations that peptide binding is mediated by ionic interactions with the loop following the binding site, referred to here as the loop. These contacts are found with the 9-mer peptide, while the shorter 5-mer unbinds spontaneously after a few tens of ns. Recent experimental results on several PDZ domains support our interpretation [23], [30]. Strong differences between short and long peptides were found for negatively charged loops (e.g. MAGI1-PDZ2) [30]. Peptide-loop contacts are dynamic, where multiple specific interactions interconvert on a fast time scale of tens of ns (i.e. much faster than unbinding [22], [31]). Such dynamic interactions are likely to characterize several other PDZ domains. Further calculations on another member of the PDZ family, the DLG1-PDZ2, which is characterized by a different loop, support our hypothesis. Moreover, unresolved side chains away from the C-terminus are often found in other PDZ-ligand X-ray structures (see examples in Table S2), indicating that these side chains can adopt multiple conformations. We note that the presence of positively charged residues downstream of the fourth C-terminal positions of PDZ peptide ligands is well attested by recent experimental specificity profiles [7]. These charged residues are not necessarily always at the same positions, even within ligands of the same domain [30]. This is likely so because the peptide is flexible at these positions (as shown in Fig. 2). Consistently, loops display a clear over-representation of negatively charged residues compared to other regions in PDZ domains: 11.6% of D/E in entire PDZ domains, 15.2% for D/E in loops (according to the Fisher's test the probability to have this difference by chance is as low as , see Methods). Many of these residues on the loop provide clusters of negatively charged side chains that are ideally suited to recruit ligands with positive charges at any position between −4 and −7.
Second, we propose a mechanistic explanation for the microscopic origin of the binding entropic penalty in absence of the extra-domain helix of PSD95-PDZ3. In the apo form, the helix plays a crucial role in stabilizing both the carboxylate binding loop and the loop. Hence, these two loops are more flexible in the helix truncated domain. In this case, the peptide quenches the two regions upon binding, resulting in the observed entropic penalty. This quench does not take place when the extra-domain helix is present. Our findings suggest that extra-domain regions might play a more important role than mere linkers between functional domains [16], reiterating that the reductionist approach that protein domains can be studied in isolation should be always validated. This is especially important because several segments adjacent to domains show little sequence specificity (and thus are often not included in domain definition), although they adopt well-defined secondary structures such as the in the third PDZ domain of PSD95.
Third, analysis of 258 human PDZ domains as well as MD simulations of single-mutants allowed for the identification of an amino acid at the beginning of the loop, VAL in PSD95, that correlates with the presence of the extra-domain helix in other PDZ domains. Prediction of helical propensities at positions following the C-terminus of the domain showed enhanced probability for those domains presenting bulkier aliphatic side chains other than alanine at that position. This analysis suggests that a binding mechanism, indirectly involving the extra domain helix as in PSD95-PDZ3, might be relevant for a significant portion of the PDZ domain family.
Molecular dynamics simulations were performed using the GROMACS implementation [32] of the CHARMM27 force field [33], [34] at constant temperature and pressure with reference values equal to 300 K and 1 atm, respectively. The use of hydrogen virtual sites and fixed covalent bonds allowed a 4 fs integration time-step [35]. All systems were solvated in a dodecahedric box with an average of roughly 5000 tip3p water molecules (see Table S1 for details of each simulation setup). In the case of PDZ3, the system was equilibrated from the deposited X-ray structures 1BE9 and 1BFE [1] for the bound and apo forms, respectively, using residues 306–402 for the WT and 306–395 for . The PDZ2 starting structure is 2I0L [13] (from DLG1/SAP97). Each molecular setup was sampled by four independent runs of approximately 200 ns each for a total of (Table S1). The first 50 ns of each trajectory were neglected in the analysis to reduce the bias from the starting configuration. Snapshots were saved every ps. The peptide N-terminus was neutralized in all cases, except CRIPT5*. The sequences of the 9-mer peptides are -TKNYKQTSV-COOH and -LQRRRETQV-COOH for PDZ3 and PDZ2, respectively. The first 5 peptide residues (i.e., positions from −4 to −8) as well as mutations at position 328 and the truncation of the extra domain helix were modeled using PyMol [36]. For each run, backbone RMSF values were calculated per residue as an average over the atoms C, and N. Final RMSF values were averaged over the four runs. Molecular trajectories were analyzed with the programs WORDOM [37], [38] and GROMACS [39]. Hydrogen bonds were determined based on cutoffs for the angle Acceptor - Donor - Hydrogen () and the distance Donor - Acceptor (3.6 Å). Ionic interactions are considered to occur when the two last carbons before the charged atoms are closer than 5 Å.
Structural analysis
Each protein-ligand snapshot was labeled by a four-digits code. The first three digits describe the peptide-loop interactions, e.g. "110". The last digit represents an id, encoding the peptide structural conformation (i.e., the internal degrees of freedom). The latter was obtained by running a leader-based cluster-analysis on the ligand backbone (atoms C, and N) with a 2 Å cutoff, using the program WORDOM [37], [38]. This digit distinguishes between different peptide conformations characterized by the same contacts with the loop. Each four-digit string represents a microstate of the protein-ligand complex. This decomposition is used to build a conformation-space-network [40]–[42], where each microstate is a node and a link between two nodes is placed if there is a direct transition between them during the MD simulation. Basins of attraction are defined using a gradient-cluster analysis [43], [44], where multiple microstates are lumped together if they interconvert rapidly. Each gradient-cluster represents a metastable configuration, which can contain heterogeneous peptide-loop contacts. Connectivity between these metastable configurations is represented as a coarse-grained network as shown in Fig. 3 (see also Fig. S2 in the Supp. Mat.). The gradient-cluster algorithm is freely available in the program PYNORAMIX (GPL license, available at the website raolab.com).
Predictions of free-energy differences upon mutations were done with FoldX using the BuildModel option after properly repairing the structures with the RepairPDB command [27]. The initial structure (PDB 1BFE) was first minimized with GROMACS in explicit water. This structure was originally crystallized with an ILE at position 328. We mutated it both to VAL (WT) and ALA to compute the free-energy differences.
Human PDZ domain sequence analysis
The set of all human PDZ domains was retrieved from PFAM [45] and SMART [46] databases. A first multiple sequence alignment was generated with MUSCLE [47]. The alignment was manually curated, removing PDZ domains that could not be unambiguously aligned (most of them are unconventional PDZ domains). This resulted in a total number of 258 PDZ domains (see Table S3). The loop was mapped by homology starting form the structure of PSD95-PDZ3. Several PDZ domains are close paralogs, and this can result in strong biases when computing frequencies or correlation patterns. To account for this effect, we always grouped paralogs together (see Table S4). Groups of paralogs were defined using a cut-off of 50% on the sequence identity. The contribution of each member of a group was weighted by the inverse of the group size. For instance, to compute the amino acid frequency at a given position, residues from a group of 5 paralogs only contributed 1/5 each to the total frequencies. The helical propensity of C-terminal extensions of PDZ domains was computed with PSIPRED [28] for up to 20 residues downstream of the domains. If the protein C-terminus was reached before the 20 residues, a helix propensity of 0 was used. Here again, the contribution of paralogs was weighted to prevent purely phylogenetic correlations. P-values were computed by reshuffling the amino acid composition at position 328 in all PDZ domains of Table S3. The Fisher's test was used to compute the probability to have a given number of negative residues within all loop residues, knowing the total number of negative residues within the sequences of all PDZ domains [48].
Supporting Information
Figure S1.
MD simulation time series of the PDZ3 complexed with the 5-mer CRIPT peptide. A rebinding event of the 5-mer CRIPT peptide to PDZ3 is highlighted in grey. (Top) Distance between the 5-mer peptide side chain nitrogen of and the loop of . (Bottom) Backbone RMSD from the X-ray structure of peptide residues 0:−4 for the 5-mer and the 9-mer peptides are shown in blue and black, respectively. The longer peptide stays tightly bound for the whole simulation time (). The short peptide immediately goes into a partially unbound state (), unbinding completely after roughly 110 ns. At 50 ns a rebinding event occurs (grey areas). During this event the two charged side chains of and come closer, suggesting that the rebinding process is mediated by this ionic interaction.
Figure S2.
CRIPT peptide affinity scheme for PSD95-PDZ3. (Top) Schematic representation of the domain-peptide complex. Experimental binding affinities from Ref. [17] are reported (no affinities are found for , indicated with a *). (Middle) Transition network between the different peptide binding modes (see main text for details). Pie charts surfaces indicate the population ratio between the different configurations. Interactions involving exclusively or are indicated in pink and red, respectively. In dark red both lysines are engaged with the loop, white for no interactions. (Bottom) The distribution of the total number of canonical interactions for each complex. We monitored three key contacts. They are the hydrogen bond between and the side chain oxygen of ; the hydrogen bond between the hydroxyl oxygen of and (a milestone for PDZ specificity); the hydrogen bond between and the side chain oxygen of .
Figure S3.
Amino acid frequencies at position 328 in human PDZ domains. Contribution of paralog domains have been weighted as described in Methods.
Figure S4.
Conservation of the loop and extra-domain alpha helix in PSD95-PDZ3 orthologs. Green shading corresponds to biochemically similar side chains. Orange shading corresponds to non-conserved residues.
Table S1.
Details of the simulations performed.
Table S2.
Examples of PDZ structures in complex with ligands with unresolved residues between position −4 and −7.
Table S3.
Manually curated multiple sequence alignment of the 258 human PDZ domains used in this work.
Table S4.
Clusters of human PDZ paralogs.
We thank Diego Prada-Gracia for interesting discussions; Mikael Akke for elucidations on NMR spectroscopy; Katja Luck and Gilles Travé for sharing their manuscript before publication [30].
Author Contributions
Conceived and designed the experiments: SM FR. Performed the experiments: SM DG FR. Analyzed the data: SM DG FR. Contributed reagents/materials/analysis tools: SM DG FR. Wrote the paper: SM DG FR.
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text:
TDS u/s 194I - assessee paid by the assessee to MMRDA for acquiring lease hold rights and additional FSI for the leased plot - CIT(A) held the payment was not in the nature of rent as defined in Section 194I of the IT Act, and is not liable for TDS - Held that:- A careful reading of the said lease deed transpires that the premium is not paid under a lease but is paid as a price for obtaining the lease, hence it precedes the grant of lease. Therefore, by any stretch of imagination, it cannot be e .....
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BKC. The Development Control Regulations for BKC specify the permissible FSI. Pursuant to such provisions, the assessee became entitled for additional FSI and has further acquired/purchased the additional built up area for construction of additional area on the aforesaid plot. Thus the assessee has made payment to MMRD under Development Control for acquiring leasehold land and additional built up area.
The decision of the Hon'ble Jurisdictional High Court in the case of Khimline Pumps Ltd .....
M/2014, CO No. 116/MUM/2015 - Dated:- 17-8-2016 - SHRI B.R.BASKARAN (AM) AND SHRI RAM LAL NEGI (JM) For The Appellant : Shri. Dr. Darshi Suman Ratnam For The Respondent : Shri. M.P. Lohia & Nikhil Tiwari ORDER PER RAM LAL NEGI, JM The present appeal and Cross Objection have been preferred by the Revenue and the assessee respectively against order dated 14/03/2014 passed by the Ld. CIT(A)-37, Mumbai for the Asst. year 2010-11. Since the appeal and cross objection pertain to the same assessee .....
short The Act ) determining the total income of ₹ 1,77,04,05,282/- after inter alia making disallowance of ₹ 51,32,63,248/- towards lease rent payment made to MMRDA. The assessee challenged the assessment order before the CIT(A). The Ld. CIT(A) allowed this ground of appeal of the assessee and deleted the disallowance made u/s 40(a)(ia) of the Act. Against the said order the revenue is in appeal before the Tribunal. 2. The revenue has challenged the impugned order dated 14/03/2014 p .....
appreciating that, the amended definition of rent in Explanation to section 1941 w.e.f. 13/07/2006 is comprehensive and includes any payment by whatever name called under any lease, sublease, tenancy or any other arrangement. 3. At the outset, the Ld. Counsel for the assessee submitted that the issues involved in the present appeal have been decided in favour of the assessee and against the revenue by the ITAT Mumbai in assessee s own cases ITA No 1734/Mum/2012 for the A.Y. 2008-09 and ITA No 44 .....
ed that since each and every case is required to be decided on its own merit, the AO has rightly made the disallowance in question and the Ld. CIT has wrongly allowed the appeal of the assessee. 5. We have heard the rival submissions and perused the documents carefully in the light of the respective contentions of the parties. We have noticed that the coordinate Bench of the Tribunal has decided the identical issues in favour of the assessee in assessee s own cases referred above, by following t .....
(MMRDA) for additional built up area. According to AO such payment made by the assessee is in the nature of rent for the use of land, therefore, the assessee was required to tax under section 194I of the Act. Accordingly, vide order dated 29/3/2011 passed under section 201(1) and 201(1A) the assessee has been fastened with the tax liability as under: Tax under section 201 - ₹ 58,95,94,618/- Interest U/s. 201(1A) - ₹ 23,58,37,848/- Ld. CIT(A) has deleted such addition made by the AO. .....
of order passed in the case of ITO vs. M/s.Wadhawa & Associates Realtors Pvt. Ltd.(supra) is as under: 9. We have considered the rival submissions, perused the order of the lower authorities and the material evidence brought on record in the form of paper Book and the judicial decisions relied upon by the rival parties. The entire grievance revolves around the premium paid by the assessee to M/s. MMRDA Ltd. for the leasehold rights acquired by the assessee through the lease deed dt. November .....
d as exhibited from page-1 to 42 of the Paper Book. A careful reading of the said lease deed transpires that the premium is not paid under a lease but is paid as a price for obtaining the lease, hence it precedes the grant of lease. Therefore, by any stretch of imagination, it cannot be equated with the rent which is paid periodically. A perusal of the records further show that the payment to MMRD is also for additional built up are and also for granting free of FSI area, such payment cannot be .....
p area for construction of additional area on the aforesaid plot. Thus the assessee has made payment to MMRD under Development Control for acquiring leasehold land and additional built up area. The decisions of the Tribunal in the case of M/s. National Stock Exchange (supra) and Mukund Ltd (supra) have been well discussed by the Ld. CIT(A) is his order. The decision of the Hon ble Jurisdictional High Court in the case of Khimline Pumps Ltd. (supra) squarely and directly apply on the facts of the .....
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