neisseria gonorrheae ( ng ) and neisseria 
 meningitides are gram - negative bacterial pathogens responsible 
 for gonorrhea and meningococcal meningitis , respectively . for these 
 bacteria , phagocytosis , cellular invasion , is induced by the binding 
 of  opacity - associated  ( opa ) proteins to host receptors . opa proteins are eight - stranded -barrel integral outer membrane 
 proteins with four extracellular loops ( figure 1a ) . high sequence diversity is observed in regions of the extracellular 
 loops of opa variants ( table s1 , supporting information ) , which is predominantly a result of recombination events between 
 genes of the same isolate ( 70% ) and import of genes from other isolates 
 ( 16% ) . the three variable regions within 
 the extracellular loops , hypervariable 1 and 2 ( hv1 and hv2 ) and the 
 semivariable ( sv ) regions , engage host receptors to induce phagocytosis 
 and determine the specific host receptors engaged . the regions 
 vary in length and do not comprise the entire extracellular loop : 
 sv is 310 amino acids , hv1 is 2431 amino acids , and 
 hv2 is 4551 amino acids . there are 26 sv , 96 hv1 , and 127 
 hv2 different sequences in the 338 distinct opa alleles sequenced 
 ( http://www.neisseria.org ) . this sequence diversity likely 
 plays a beneficial role in helping neisseria to evade host immune 
 responses ; however , it poses a challenge 
 in that highly varied loop sequences must engage a common set of receptors 
 to mediate cellular invasion . the determinants of opa  receptor 
 interactions are of the utmost importance for understanding neisserial 
 pathogenesis and the innate immune response . opa proteins 
 also provide a means of foreign body cellular entry through specific 
 human receptors that can be exploited synthetically for pharmaceutical 
 and technological purposes . opahs bind to heparansulfate proteoglycans 
 ( hspgs ) directly and indirectly to integrin receptors via an hspg - mediated 
 interaction . the more abundant class , opacea , bind to the 
 nonglycosylated face of the carcinoembryonic antigen - related cellular 
 adhesion molecule ( ceacam ) ig n - domain . although all ceacam receptors contain this domain , opa proteins 
 only bind to ceacam1 , 3 , 5 , and 6 , and most selectively bind to only 
 a subset . the ceacam n - domain residues that interact with opacea have been identified : y34 and i91 are essential for all opacea interactions , and an additional seven nearby residues are 
 implicated in binding depending on the particular opacea . the specificity - determining residues 
 on opacea are predominantly in the hv1 and 2 regions , and 
 the hv sequences are concomitant : chimeric opa proteins with an hv1 
 and hv2 region from two opa proteins that bind the same receptor do 
 not bind . toward understanding the 
 molecular recognition required to gain 
 entry into human cells , we report the structure of opa60 , which binds ceacam1 , 3 , 5 , and 6 . structure 
 determination of membrane proteins is challenging and even more so 
 for proteins that have large portions of both soluble and membrane - embedded 
 regions . thus , opa proteins presented some methodological obstacles 
 in both nmr resonance assignments and 
 structure calculation and refinement . to overcome these obstacles , 
 we employed a hybrid method that used the restraints determined with 
 solution nuclear magnetic resonance ( nmr ) spectroscopy in detergent 
 micelles in conjunction with molecular dynamics ( md ) simulations in 
 a lipid bilayer . this approach preserved the structural features that 
 were well - determined spectroscopically but employed a more physical 
 sampling method ( molecular dynamics versus simulated annealing ) and 
 more detailed treatment of solvation and electrostatics to better 
 define regions that either are flexible or remained underdetermined 
 from the spectroscopic data alone . opa60 is a canonical 
 eight - stranded -barrel with extensive ionic interactions inside 
 the barrel . three of the extracellular loops are longer than those 
 found in any -barrel structure previously determined . the hv 
 regions within these loops are dynamic on the nanosecond time scale 
 and are predominantly disordered . however , the loops are compact and 
 interact with each other weakly such that long - lived specific intraloop 
 interactions are not observed . the diverse and dynamic nature of the 
 loop structural ensemble is likely required for highly variable opa 
 loop sequences to bind a common receptor and also for a single opa 
 protein to bind a variety of host receptors . ( a ) opa60 is an eight - stranded 
 -barrel with long extracellular 
 loops ( 60% of the protein ) . charged residues within the -barrel 
 region are colored green and in the periplasmic turns colored blue . aromatic 
 residues located near the headgroup region of the bilayer are colored 
 yellow . the semivariable region ( sv ) is colored orange , and the hypervariable 
 ( hv ) regions 1 and 2 are colored red . residues in the loops that have 
 nmr assignments have gray circles and regions demonstrating transient 
 helices in the md simulations have black outlined font . ( b ) the n , h trosy - hsqc is labeled with the nmr backbone assignment 
 for opa60 in dpc micelles . ( c ) sample strips from the n - noesy spectrum indicating intra- ( solid lines ) and interstrand 
 ( dashed lines ) noes observed . residues in -strand 4 ( black ) , 
 -strand 3 ( blue ) and -strand 5 ( red ) are labeled . ( d ) 
 nmr n , h trosy - hsqc spectra of opa60 in nanodiscs containing dmpc lipids with peaks labeled with the 
 nmr backbone assignment . the spectra shown were recorded at 800 mhz 
 and at 40 c ( b , c , and d ) or 10 c to resolve only loop 
 resonances ( d ) . all nmr samples were deuterated with labile protons 
 back exchanged and the protein concentrations were 750 m 
 for dodecylphosphocholine samples and 500 m for nanodiscs 
 preparations . the gene for 
 opa60 with n- and c - terminal fusion tags was subcloned 
 into pet28b from the orginal pex vector provided ( martine bos , ultrech 
 university ) and transformed into bl21(de3 ) e. coli . cells were grown in d2o ( 99.8% ) minimal media containing 
 4 g / l c(99%)-glucose and 1 g / l n(99%)-ammonium 
 chloride ( cambridge isotopes laboratory ) at 310 k until an od600 of 0.8 expression was induced with 1 mm isopropyl--thio - d - galactoside for 8 h. cells were lysed after resuspension in 
 50 mm tris  hcl and 150 mm nacl ( lysis buffer ) . cell debris 
 from the lysate was removed via centrifugation at 12000 g for 30 min . the pellet was resuspended in lysis buffer with the 
 addition of 8 m urea overnight and centrifuged again at 12000 g for 30 min . the soluble fraction was added to a co - immobilized metal affinity chromatography column and washed 
 with 15 cv of 20 mm sodium phosphate , ph 7.8 , 150 mm nacl , 20 mm imidazole , 
 8 m urea followed by a 5 cv elution ( 20 mm sodium phosphate , ph 7.0 , 
 150 mm nacal , 680 mm imidazole ) . the eluted protein fraction was concentrated 
 to 200 m and rapidly diluted 20-fold with 20 mm tris hcl , 
 ph 8.0 , 500 mm nacl , and 4.5 mm n - dodecylphosphocholine 
 ( dpc ; anatrace ) . after 5 days of room temperature incubation , the 
 protein was fully folded as assessed with sds  page gel shift 
 analysis . the sample was then concentrated and dialyzed against 3 
  4 l of 20 mm sodium phosphate , ph 6.2 , and 150 mm nacl for 
 1 h each . final nmr samples were concentrated to 400800 m 
 and contained 110150 mm dodecylphosphocholine ( dpc ) as measured 
 by comparing sample detergent intensities with standard concentrations . opa60 was reconstituted into nanodisks according to established 
 protocols using plasmid for msp1d1h5 generously provided 
 by gerhard wagner ( harvard university ) . msp1d1h5 was purified 
 and assembled in 20 mm tris / hcl ph 7.5 , 100 mm nacl , and 5 mm edta 
 buffer with the appropriate amount of dry lipid / detergent to obtain 
 a mixture of msp1d1h5/dmpc / sodium cholate with a molar ratio 
 of 1:50:100 . opa60 refolded in dpc was added to the mixture , 
 and the opa60/msp1d1h5 ratio was adjusted to 1:4 . the mixture was incubated at 4 c for 1 h , and detergent was 
 removed with 0.5 g of washed biobeads sm-2 ( biorad ) per ml 
 of assembly mixture . h. biobeads were pelleted by centrifugation , and the 
 decanted supernatant was concentrated and purified on a superdex 200 
 gel filtration column equilibrated with 20 mm sodium phosphate , ph 
 6.5 , 50 mm nacl , and 5 mm edta . fractions corresponding to the main 
 peak were pooled and concentrated using an amicon centrifugal filter 
 unit of 30 kda mwco ( millipore ) . the nmr sample consisted of 0.5 
 mm h , n opa60 in msp1d1h5 
 nanodiscs with d54 - 1,2-dimyristoyl - sn - glycero-3-phosphocholine 
 ( dmpc ; avanti polar lipids ) , in gel filtration buffer supplemented 
 with 10% ( v / v ) d2o . nmr spectra were collected on bruker 
 avance spectrometers operating at proton frequencies of 600 and 800 
 mhz equipped with bruker 5 mm txi cryoprobes and recorded at 313 k. 
 spectra were processed with topspin and assigned using cara ( cara.nmr.ch ) . 
 the assignment strategy for opa60 is published and mapped onto the n , h - trosy - hsqc in figure s1 ( supporting information ) . through these strategies , complete nitrogen , hydrogen , c , 
 c , and co resonances were assigned for residues 114 , 
 2931 , 5171 , 9597 , 109110 , 118140 , 
 157 , 159178 , 190212 , 231 , and 233238 ( figure 1b ) with only c , c , and co resonances 
 for the seven assigned prolines and a lack of c assignment 
 for two additional resonances ( y71 and i97 ) . further , the entire side 
 chain carbon and hydrogen assignments were obtained for residues 159178 
 through tocsy and cosy assignment using a corresponding synthetic 
 peptide . additionally , nine aromatic side chain protons ( y10 , f62 , 
 w65 , f125 , f131 , y134 , f200 , y236 , and f238 ) were observed due to 
 incomplete protein deuteration and dynamics of the side chain and 
 assigned using the n - noesy spectrum . resonance assignments 
 were achieved for 92% of the -barrel region ( as defined by 
 the md refined structure ) and 27% of the extracellular loops . the 
 nspa structure was not used for the assignment or identification of 
 restraints . the talos+ 
 program was used 
 to obtain backbone dihedral angle restraints . assigned noe peak heights 
 were measured and binned into strong , medium , or weak interactions . 
 observed inter--strand noes are schematically indicated in 
 figure 1a . thirty - one additional noes between 
 backbone hn protons and aromatic side chain protons were also included . these were assigned upper limits of 3.5 , 5.0 , and 6.5  . in most 
 cases , hydrogen bonding partners could be unambiguously assigned based 
 on noe patterns ( a representative strip is shown in figure 1c ) , and two distance restraints were used with upper 
 limits of 2.5 and 3.5  for hno and no , 
 respectively . additionally , planar restraints ( 32   4 
  as a square potential ) were used to represent the lipid bilayer . the extracellular loops are very long , and without the bilayer restraint 
 they sampled conformers that would be embedded into or transverse 
 the bilayer ( figure s1 , supporting information ) . the restraint distance was chosen on the basis of ( 1 ) the hydrophobic 
 thickness of porb a -barrel membrane protein from ng for which 
 there is a crystal structure and ( 2 ) 
 the residues that were observed to have noes with detergent headgroup 
 choline protons ( figure s2 , supporting information ) . starting at 3000 k , 5000 steps of high - temperature annealing was 
 used to fold the initial extended structure . twenty of the lowest 
 overall violation energies of the 300 calculated structures with selected 
 for further md simulations . all simulations were 
 performed using 
 gromacs 4.5 and the charmm36 forcefield for protein and lipid interactions . as detailed 
 below , distance and dihedral restraints derived from the nmr data 
 and used in xplor simulated annealing runs were enforced throughout 
 the molecular dynamics simulations . simulations were run under npt 
 conditions using the velocity - rescaling thermostat at 300 k with a time - constant of 0.1 ps and semi - isotropic 
 pressure coupling using the parrinello all covalent bonds were constrained 
 using lincs , and long - range electrostatics 
 were computed every step using particle mesh ewald ( pme ) . a lipid bilayer of 512 dmpc molecules 
 ( bilayer thickness , 34   1  ; hydrophobic thickness , 2326  ) was constructed using the charmm - gui membrane builder tool and solvated with approximately 40000 tip3p 
 waters . ions were added to obtain a system with 150 mm nacl and no 
 net charge . the dmpc bilayer was equilibrated prior to protein insertion 
 with a resulting area per lipid headgroup of 0.60 nm , 
 close to the experimentally determined value ( 0.606  0.005 nm ) . the system dimensions were 
 approximately 12.5 nm ( sides ) and 11 nm ( height ) . each of the 
 20 lowest energy structures from xplor simulated annealing 
 was independently inserted in the equilibrated membrane using the 
 gromacs tool g_membed , removing approximately 
 1520 lipids in the process . each system was then energy minimized for 1000 
 steps using the steepest descents method . twenty production runs , 
 one per protein structure , were then carried out for 100 ns using 
 a time step of 2 fs . noe - based distance restraints and 
 talos+-derived dihedral restraints as used in the xplor simulated 
 annealing calculations were imposed using spring potentials with force 
 constants of 1000 kj / mol / nm and 1000 kj / mol / rad , respectively . to perform the c density analysis , all 
 trajectories were 
 aligned to a single consistent reference structure using a rigid - body 
 fit where the objective function was calculated only on the  
 sheet and turn residues . then the density was calculated on a 3d - grid 
 with the mdanalysis toolkit , using a 
 grid - spacing of 0.1 nm . clustering was performed using 
 the gromacs tool g_cluster , which 
 was extended to include the k - means algorithm . the 20 most - populated , and therefore lowest free energy clusters , 
 were selected , and the minimum energy structure from each was reported 
 to form the hybrid refinement ensemble . the c rmsd within each cluster ranged from 1.52 
 to 4.22  and 1.87 to 5.33  for the entire protein and 
 the extracellular loops , respectively ( table s2 , supporting information ) . the rmsd for the extracellular loops 
 between clusters is much greater with the pairwise rmsd for the minimum 
 energy structures ranging from 5.60 to 29.3  ( table s3 , supporting information ) . only contacts greater 
 than 1.5 kt estimated free energy difference between the md and xplor 
 structures additional analysis of contact between 
 the hv1 and hv2 loops was 
 performed by generating contact maps between these residues , where 
 a contact was defined using a 6 interatomic distance cutoff . contact probabilities and lifetimes were computed using these contact 
 maps , and highly contacting structures ( defined as > 50 simultaneous 
 contacts ) were further analyzed via agglomerative single - linkage clustering 
 using euclidean distance on the hv1-hv2 contact maps , yielding 10 
 clusters of hv1hv2 contacts . side chain 
 restraints were not included 
 in the md / nmr hybrid refinement . xplor ensemble without side chain 
 noes had a mean global backbone rmsd for the -sheet residues 
 of 1.04  0.15  . chemical shifts were 
 calculated on all snapshots in the md simulations and for the xplor 
 structures using the sparta+ software . the calculated shifts were then averaged for each atom , and the 
 average values compared to the experimentally determined shifts ( which 
 were corrected for deuterium and trosy induced shifts ) , where existing . 
 in all cases , the errors reported are those used by sparta+ ( 0.92 
 ppm for c and 0.49 ppm for hn , respectively ) . relaxation rates 
 were measured using 
 two - dimensional n  h trosy - based experiments 
 recorded at 600 mhz and 313 k. nmr data were processed and fit with 
 nmrpipe . h 
 bond vector ci(t ) = < i ( 0 ) , i ( t ) > , 
 averaged across all trajectories , where i is the 
 n  h bond vector for the ith residue . ( a ) 
 -barrel and periplasmic turns are colored black ; extracellular 
 loop 1 , green ; loop 2 , blue ; loop 3 , red ; loop 4 , magenta . ( b ) differences in carbon chemical shifts 
 compared to random coil values are plotted ; ( c  c ) = /3(ci1 + ci data for 
 turns are colored red , for -strands blue , for loops green , 
 and for the n - terminus gray . the chemical shifts have been deposited 
 in the biomagresbank under the bmrb accession no . atomic coordinates 
 for the xplor and md / xplor refined 20 conformers representing the 
 structure of opa60 have been deposited in the pdb ( pdb 
 i d : 2mlh and 2maf , respectively ) . opa60 is 
 an eight - stranded 
 -barrel with four extracellular loops ( figures 1a and 2a ) . the combination of a stable , 
 membrane - inserted -barrel domain and long unstructured loops 
 complicated assignment and structure determination . strategies for 
 the assignment of the protein included trypsin cleavage , peptide synthesis , 
 and assignment at various temperatures . hn 
 noes , h - bond restraints , and backbone dihedrals calculated with talos 
 ( table 1 ) . the backbone rmsd of the barrel 
 region for the 20 lowest energy structures is 0.96  ( table 1 and figure 2a ) . the structure 
 calculations were complicated by the long unstructured loops ; in the 
 initial calculation , extracellular loops adopted unreasonable conformations 
 that spanned the membrane embedded region with excursions to the periplasmic 
 side of the protein ( figure s1 , supporting information ) . to address this problem , planar restraints were introduced at 
 a distance of 32   4  ( see the experimental section for details ) . most of loops 1 , 
 2 , and 4 were not assigned ( figure 2b ) because 
 the resonances were not observed ( although spectral overlap contributed ) . lowering the temperature broadened -barrel 
 and some loop peaks beyond detection simplifying the spectra to only 
 the most dynamic regions of the loops and facilitating 27 loop resonances 
 to be assigned . to further assign the functionally important hv2 region , a synthetic 20 amino acid peptide was synthesized and had nearly complete 
 spectral overlap with the full - length protein allowing 17 loop resonances 
 to be assigned . there are two likely phenomena that contribute 
 to the line broadening of the resonances that are not observed : ( i ) 
 conformational exchange and ( ii ) structural heterogeneity . the former 
 was speculated to contribute to the lack of assignments in ompx and ompa in dihexanoylphosphatidylcholine 
 ( dhpc ) and dpc micelles , respectively . the missing resonances ( approximately 
 half of the extracellular loops ) corresponded to residues that connect 
 the ordered -barrel and the flexible central region of the 
 extracellular loops . some of the missing loop resonances of ompx were 
 resolved when the micelle was replaced with nanodiscs containing dmpc 
 and 1,2-dimyristoyl - sn - glycero-3-phosphoglycerol 
 ( dmpg ) . to better refine the structure , each of the 20 structures of the 
 nmr ensemble was subjected to molecular dynamics simulations in a 
 dmpc lipid bilayer . an interval of 100 ns of simulation was selected 
 for the structure refinement because t1 and t2 relaxation data indicated 
 the hv regions were dynamic on the nanosecond time scale . in addition , 
 the chemical shifts and cd indicated the hv and extracellular loops , 
 respectively , are random coil . although the solution nmr structure 
 was determined in dpc , the simulations were sought in a more biologically 
 relevant membrane environment . there are several pieces of evidence 
 that suggest opa60 has the same structure in dmpc as in 
 dpc . cd spectra of opa60 in dpc and dmpc small unilamellar 
 vesicles ( liposomes ) indicate the protein structure is approximately 
 50% random coil 50% -strand ( figure s3 , supporting information ) . in addition , opa60 in 
 nanodiscs with dmpc have -barrel chemical shifts that are superimposable 
 with the dpc spectra ; however , a few barrel resonances corresponding 
 to residues on strands 3 , 6 , and 8 are missing in the nanodisc spectrum 
 ( figure 1d ) . to further refine the solution 
 nmr structure , 100-ns md simulations were performed on each of the 
 20 lowest energy nmr structures embedded in a dmpc lipid bilayer ( bilayer 
 thickness , 34  1  ; hydrophobic 
 thickness , 2326  ) . the resulting ensemble 
 from the md simulations has a backbone rmsd of 1.19  for the 
 -barrel region ( figure 3a and table 1 ) . the md ensemble is composed of the minimum energy 
 structure from each of the 20 lowest free energy clusters and captures 
 2937 of the 4000 snapshots ( table 1 ) . the c 
 rmsd within each cluster ranged from 1.524.22  and 1.875.33 
  for the entire protein and the extracellular loops , respectively 
 ( table s2 , supporting information ) . the 
 rmsd for the extracellular loops between clusters is much greater 
 than within clusters , with the pairwise rmsd for the minimum - energy 
 structures ranging from 5.60 to 29.3  ( table s3 , supporting information ) . although sampling is 
 not sufficient to achieve full convergence , principal components analysis 
 of the loop conformations across all simulations yields good overlap 
 in the subspace of the two largest principal components ( figure s4 , supporting information ) , indicating that at least 
 in terms of the highest amplitude loop motions , individual simulations 
 sampled overlapping rather than isolated regions of conformation space . thus , the ensemble from the 20 lowest energy clusters represents the 
 loop structural diversity observed in the trajectories . the 
 -barrel of opa60 is similar in sequence to nspa ( figure s5 , supporting information ) ; however , the overall fold from the md / nmr refinement is most similar 
 to ompa and ompx ( table s4 , supporting information ) . similar to the 12 eight - stranded -barrels deposited in 
 the protein data bank , the strands have a right - handed twist ( figure 3a ) and an aromatic belt around the circumference 
 of the barrel at the lipid headgroup regions ( figure s6 , supporting information ) . a significant ionic network exists inside the barrel ( figure s7 , supporting information ) ; however , there is no 
 observable pore through the length of the barrel . the ionic 
 residues on the extracellular side of the barrel ( figure 
 s7 , supporting information ) are excluded 
 from solvent in many of the clusters , and those on the periplasmic 
 surface ( figure s7 , supporting information ) are accessible to solvent in all the clusters . this ionic network 
 may contribute to the significant stability observed for opa60 ; the barrel remains intact after cleavage with trypsin and boiling 
 in sds loading buffer . basic residues ( figure s8 , supporting information ) are clustered in the extracellular loops near the barrel domain . these residues may interact with the negatively charged lipooligosaccharide 
 outer leaflet of the outer membrane as was observed for fhua and one of the lps interactions identified with 
 oprh . however , specific interactions 
 between the barrel and lps were not detected by chemical shift perturbation 
 when lps ( los , which is in the outer leaflet of the neisseria outer 
 membrane , is not commercially available ) was titrated into the opa60dpc micelle ( data not shown ) . colored by density ( probability of a c 
 atom within an  grid cell ) from yellow ( 2  
 10 ) to blue . the gray planes show the average 
 positions of the phosphorus atoms in the lipid head groups . contact map for the ensemble calculated with xplor in detergent ( a ) 
 and for the md in lipid ( b ) . the contact map is rendered as a contour 
 plot of contact probability , with evenly spaced contours from 10% 
 to 100% contact probability in each ensemble colored from dark blue 
 to dark red . contacts 
 were defined as two atoms from respective residues approaching within 
 5  . comparison of the amide proton ( c ) and c ( d ) chemical 
 shifts calculated from the xplor ensemble and the md ensemble . positive 
 values indicate the chemical shifts calculated from the md ensemble 
 agree better with the observed chemical shifts , and negative values 
 indicate chemical shifts calculated from the xplor ensemble agree 
 better with the observed chemical shifts . the md - refined 
 ensemble has several more inter - residue contacts than the xplor refined 
 ensemble ( figure 4a , b ) . these new contacts 
 are primarily intraloop : 11 are across the periplasmic side of the 
 -barrel and 17 extend the strands on the extracellular side 
 ( only the 76 contacts that represented a greater than 1.5 kt estimated 
 free energy difference between the md and xplor structures were considered ) . consequently , the most probable loop conformations in the md refined 
 structures are much more compact than the xplor refined structures 
 ( figure 3a ) with most of the loop density above 
 the -barrel ( figure 3b ) . this decrease 
 in loop volume can be quantified via the protein radius of gyration , 
 which decreases from the initial structure over the course of 19 of 
 the 20 trajectories ( figure s9 , supporting information ) . the accuracy of the structural representation of the xplor 
 and md refined ensembles can be evaluated by comparing chemical shifts 
 calculated from the ensemble structures via semiempirical shift prediction 
 methods against the experimental chemical shifts ( exp ) . parts c and d of figure 4 compare the deviations between the experimentally 
 measured values ( exp ) of c and hn opa60 chemical shifts and the xplor and md predictions . most residues do not show differences between 
 xplor and md larger than the sparta+ 
 reported prediction accuracy . however , for carbon shifts , which primarily 
 depend on backbone dihedral angles , the md predictions agreed better 
 with the experimental shifts indicating that the md ensemble is an 
 accurate representation of the observed opa60 structure . the -barrel provides the scaffolding 
 for the functional extracellular loops , which are disordered and sample 
 a diverse ensemble of conformers . the nmr and md dynamics data ( figure 5 ) indicate that hv2 and three extracellular loops 
 ( l1l3 ) , respectively , are dynamic on the nanosecond time scale . 
 because the opa  detergent complex has a large overall correlation 
 time , t1 values are highly sensitive to backbone nanosecond motions . opa t1 values decrease significantly at the n - terminus , periplasmic 
 turns , and extracellular loops 1 and 3 compared to the -strands 
 ( figure 5a ) , indicating these regions have 
 high amplitude motions in the nanosecond time scale . several of the 
 -strands have a general trend of increased dynamics toward 
 the n- and c - terminal ends of the strands ( although most have order 
 parameters greater than 0.85 ) , which was previously reported for other 
 -barrel membrane proteins investigated with nmr . t2 changes are much more difficult to interpret since values increase 
 with nanosecond motions and decrease with s  ms motions . 
 nonetheless , the opa60 t2 values ( figure 5a ) are consistent with the interpretation of the t1 values . thus , on the basis of the nmr data , the hv2 region of opa has a high 
 amplitude of motion on the nanosecond time scale . these nmr data are 
 consistent with those observed with md , which provides a more comprehensive 
 understanding of the motions of the loops . although sampling of loop 
 conformations was not globally converged , the rank order of backbone 
 dynamics showed good convergence . assessed at 20 ns , the spearman 
  was 0.975 between the mean autocorrelation function value 
 and the fifth percentile of sampled trajectories , while the  
 between the mean and the 95th percentile was 0.988 . a gradient is 
 observed for the md - derived backbone nh bond vector time autocorrelation 
 function for each of the three longer extracellular loops ( l1l3 ; 
 figure 5b ) , with loop regions furthest from 
 the barrel more dynamic than the regions closest to the barrel . based 
 on the md simulations , the sv , hv1 , and hv2 regions are moving within 
 the nanosecond time regime and with a high amplitude of motion . ( a ) n t1 and n t2 relaxation 
 values for opa60 plotted versus sequence 
 and secondary structure . n t1 values that are less than 
 80% ( dotted line ) of the value predicted ( 1 s ) for a 20 ns overall 
 correlation time have s values less than 
 0.85 ( e = 10 ps ) . data for turns are colored red ; 
 -strands , blue ; and loops , green . ( b ) cartoon representation 
 of opa60 with residues colored according to the n  h 
 orientational time autocorrelation function calculated from md trajectories . as might be expected from the 
 extensive nanosecond - time scale dynamics , the loops do not maintain 
 long - lived structural features . however , they do form considerable 
 intraloop contacts , which are captured more readily by the hybrid 
 refinement strategy than simulated annealing alone ( figure 4a , b and 6a ) . there are several 
 contacts within each loop that are observed in the md refined structures . 
 of the 76 contacts above 1.5 kt estimated free energy difference between 
 the xplor and xplor / md ensembles , 31 are within each of the three 
 loops ( l1l3 ) . contacts between hv1 and hv2 are of most interest 
 since they are both required to bind to ceacam receptors . common contacts 
 between the two regions are observed ( figure 6a ) ; however , these contacts have short lifetimes ( figure 6b ) . throughout the simulations these contacts are 
 frequent but short - lived . based on this observation , the 4000 snapshots 
 were reclustered based on the hv1 and hv2 regions and analyzed in 
 terms of contacts and representative structures ( figure s10 , supporting information ) . recurrent contacts were 
 observed between residues in the range 153165 ( hv1 ) and 8695 
 ( hv2 ) as well as 171180 ( hv1 ) and 9498 ( hv2 ) . not 
 surprisingly , there are several hydrophobic residues that mediate 
 these hv1hv2 interactions ( figure s10 , supporting information ) . the recurring contact patterns were 
 observed in loop conformations that were globally quite different 
 and across multiple independent md simulations from different starting 
 structures , suggesting robust formation of transient yet frequent 
 interactions . these observations are broadly consistent with the primary 
 nmr data in that long - lived interactions or persistent structure in 
 hv1 and hv2 were not observed on the basis of chemical shift ( figure 2b ) and the lack of nonsequential noes in assigned 
 regions . ( b ) average contact 
 lifetimes are plotted for each contact , with each contour line representing 
 a 5-ns lifetime increment . comparison of these panels shows that hv1hv2 
 contacts are relatively frequent but short - lived . in addition to the contacts observed , the sv , hv1 , and hv2 
 regions 
 each sample helical conformers in a small fraction of the 4000 snapshots 
 of the 20 trajectories ( figure s11 , supporting 
 information ) . other secondary structures , such as ppii and 
 310 helices , were less abundant in these regions ( figure 
 s11 , supporting information ) . the existence 
 of these lowly populated structures is difficult to probe with traditional 
 nmr methods ; however , for populated secondary structure elucidation , 
 carbon chemical shifts ( figure 2b ) are typically 
 used . for the data obtained , only a few residues in the sv and hv2 
 regions indicate -helical structure ( positive values ) , but 
 overall the values indicate the dominant population is random coil 
 which is consistent with the md results . the lack of any long - lived 
 discrete structure in the extracellular regions of opa60 is compatible with the degree of sequence variability that still 
 confers binding to host receptors ( table s1 , supporting 
 information ) . it would be surprising should such extreme variability 
 result in a single stable structure . despite the 
 structural plasticity of the extracellular loops , opa proteins must 
 still bind a common set of receptors . depending on the hypervariable 
 sequences in the extracellular loops , opa proteins bind selectively 
 to the n - domain of ceacam1 , 3 , 5 , and/or 6 but do not bind the n - domains 
 of ceacam4 , 7 , and 8 . using mutagenesis , residues y34 and i91 of ceacam n - domains ( figure s12 , supporting information ) were identified to be 
 essential for the opa - receptor interaction . several 
 other residues ( 27 , 28 , 29 , 32 , 39 , 44 , and 89 ) dictate the different 
 opa  ceacam selectivity reported ( figures s12 and s13 , supporting information ) . the total exposed surface 
 area of these identified residues is approximately 440  and is composed of both hydrophobic 
 and polar moieties , which can easily be complemented by the hydrophobic 
 and polar groups in the hv regions of opa proteins ( table s1 , supporting information ) . beyond the enthaplic 
 interactions , the dynamics and conformations of the extracellular 
 loops are important to the molecular recognition event . the extracellular 
 loops are intrinsically disordered yet are sampling a restricted volume 
 such that there are interactions between the loops on the nanosecond 
 time scale . md simulations further suggest recurrent yet transient 
 interaction patterns between specific regions of the loops . thus , 
 the loops adopt an intermediate state that is not folded but is also 
 not lacking in interactions ; the state of the loops may be best described 
 as premolten globule or both hv1 and hv2 are required ; 
 the hv regions are concomitant since chimeric opa proteins with an 
 hv1 and hv2 region from two opa proteins that bind the same receptor 
 do not bind receptor . state may be a mechanism to retain disorder yet provide conformers 
 in which hv1 and hv2 are in proximity and competent to interact with 
 ceacam . the small ceacam binding surface ( figure s13 , supporting information ) and the requirement of 
 both opa hypervariable regions suggest that a large folding event 
 of the extracellular loops is unlikely upon binding suggesting that 
 the binding mechanism is more likely conformational selection rather 
 than induced fit . however , there is a plethora of commentary on the 
 similarities and differences of these two binding mechanisms with 
 the prevailing idea that binding reactions could have elements of 
 both mechanisms . in addition , sequences are selected for function 
 not mechanism ; therefore , the mechanism of different opa proteins 
 may vary . since opa ceacam interactions differ among variants 
 and receptors , the dynamic nature of the loops maximizes the likelihood 
 that a sequence will engage the receptor by increasing conformer sampling 
 and the potential binding modes for receptor engagement . we report the structure of opa60 , a neisserial outer 
 membrane protein that induces host phagocytosis of the bacterium through 
 specific receptor interactions . this eight - stranded -barrel 
 protein possesses three extracellular loops ( greater than 34 residues ) 
 that are longer than any -barrel structures yet reported and 
 required a membrane restraint in the xplor structure calculation . to understand the structure and dynamics of the loops , we employed 
 a hybrid xplor / md refinement where nmr - derived restraints were used 
 in 20  100 ns md simulations to obtain a structure of opa60 in a dmpc membrane . the hybrid - refined structure is consistent 
 with the initial xplor structure but has an increase in loop structure 
 and compactness . although there is little secondary structure 
 evident in either the md simulations or the primary nmr data , there 
 are many short - lived contacts between the loops on the nanosecond 
 time scale due to extension of the -strands . we hypothesize 
 that the observed dynamic ensemble is critical for maximizing the 
 conformations of a highly variable region of opa to engage receptors . that is , a high degree of plasticity is required to tolerate the diverse 
 sequences in these regions and sample conformers competent to engage 
 receptors . it remains to be shown , however , whether different opa 
 variants engage a single receptor ( e.g. , ceacam1 ) via similar or different 
 loop structures . the structure of several opa variants in complex 
 with an identical receptor will elucidate whether the binding modes 
 are indeed convergent or divergent .
the structure and dynamics of opa 
 proteins , which we report herein , 
 are responsible for the receptor - mediated engulfment of neisseria 
 gonorrheae or neisseria meningitidis by 
 human cells and can offer deep understanding into the molecular recognition 
 of pathogen 
 host receptor interactions . 
 such interactions are 
 vital to understanding bacterial pathogenesis as well as the mechanism 
 of foreign body entry to a human cell , which may provide insights 
 for the development of targeted pharmaceutical delivery systems . the 
 size and dynamics of the extracellular loops of opa60 required 
 a hybrid refinement approach wherein membrane and distance restraints 
 were used to generate an initial nmr structural ensemble , 
 which was 
 then further refined using molecular dynamics in a dmpc bilayer . 
 the 
 resulting ensemble revealed that the extracellular loops , which bind 
 host receptors , occupy compact conformations , interact with each other 
 weakly , and are dynamic on the nanosecond time scale . 
 we predict that 
 this conformational sampling is critical for enabling diverse opa 
 loop sequences to engage a common set of receptors .