molecular chaperones facilitate the folding and unfolding of polypeptides and are essential for the assembly of large protein complexes ( macario and conway de macario , 2005 ) . the human complex consists of four subunits : ruvbl1 , ruvbl2 , pih1d1 , and rpap3 ( zhao et  al . , 2005 ) . ruvbl1 ( also known as pontin , ruv1 , and tip49a ) and ruvbl2 ( also known as reptin , ruv2 , and tip49b ) are essential and highly conserved atpases that belong to the adenosine triphosphatases associated with multiple activities ( aaa+ ) family . both possess atpase and protein and nucleic acid binding activity and are involved in many biological processes , including chromatin remodeling , transcription regulation , ribonucleoprotein complex biogenesis , nonsense - mediated mrna decay , and the dna damage response . in the majority of these processes , ruvbl1 and ruvbl2 function as components of larger complexes , such as the r2tp ( jha and dutta , 2009 ) . rpap3 contains tetratricopeptide repeats involved in hsp90 binding ( back et  al . , 2013 , jimnez et  al . , 2012 ) and potentially other protein - protein interactions . the last member of the r2tp complex ( pih1d1 ) contains a predicted structural domain with unknown function . evidence supports a role for the r2tp complex along with hsp90 in the assembly of a number of multisubunit molecular  machines , including small nucleolar ribonucleoproteins ( snornps ) , spliceosomal snrnp u4 , rna polymerase ii , and mtorc1 and smg1 complexes ( ahn et  al . , 2013 , nonetheless , a molecular explanation of how the r2tp complex recognizes such diverse substrates remains elusive ( machado - pinilla et  al . , we previously reported that pih1d1 binds to a casein kinase 2 ( ck2)-phosphorylated form of the cochaperone tel2 . tel2 , along with tti1 and tti2 , forms the ttt complex , which is critical for the assembly of the phosphatidylinositol 3-kinase - related kinases ( hurov et  al . , 2010 , our previous study revealed that ck2 phosphorylation of tel2 is essential for direct binding to pih1d1 , and its disruption leads to the destabilization of mtor , smg1 , and , to a lesser extent , atm , atr , and dna - pkcs ( horejs et  al . , 2010 ) . given that pih1d1 is not predicted to contain any of the known phosphopeptide binding domains , such as 14 - 3 - 3 , fha , brct , wd40 , ww , and polo box domains , it is unclear how it recognizes phosphorylated tel2 ( ptel2 ) . furthermore , whether phosphorylation - dependent binding represents a universal substrate recognition mechanism for the r2tp complex has not been previously explored . here , we demonstrate that the n - terminal pih1d1 region pih - n is a phosphopeptide binding domain structurally unrelated to  previously reported phosphopeptide binding proteins that is  required for recognition of phosphorylated substrates , whereas the c - terminal region of pih1d1 binds to the other components of the r2tp complex . the crystal structure of pih - n domain fragment bound to the phosphorylated tel2 peptide suggests that the major interacting site for the pih - n domain is a short conserved dps491dd motif , previously described as a ck2 phosphorylation site ( ahn et  al . , 2013 , the pih - n domain contains two conserved residues ( lys57 and lys64 ) that directly interact with the tel2 pser491 and are  essential for binding in  vitro and in  vivo . proteomic and in  silico screens identified several pih1d1 phosphorylation - dependent binding partners . among these , we have confirmed a direct phosphorylation - dependent interaction with human ecdysoneless ( ecd ) , previously implicated in the stabilization of the tumor  suppressor p53 , mediated by a motif identical to the tel2 dpsdd sequence . first , to gain insight into the apparent phosphopeptide binding activity of human pih1d1 and its interactions with r2tp complex components , we performed domain - mapping experiments . immunoprecipitation studies mapped the binding of phosphorylated tel2 to a region within the n - terminal 250 amino acids of pih1d1 , whereas ruvbl1 , ruvbl2 , and rpap3 interacted via a separate region in its c terminus ( figure  1a ) . additional sequence homology and limited proteolysis analysis ( figure  s1a ) , coupled with isothermal titration calorimetry ( itc ) measurements on a series of truncation mutants , was initially used to define a smaller , stable fragment ( 1180 ) that was sufficient to bind to a tel2 peptide phosphorylated on ser491 with an affinity of 3.5  m , comparable to the affinity of other phosphopeptide binding domains for their substrates ( figures 1b and s1c ; table  s1 ) ( lloyd et  al . , 2009 ) . a diphosphorylated peptide incorporating ser487 located just upstream of ser491 bound with 2- to 3-fold higher affinity , whereas binding to a peptide phosphorylated only at ser487 was substantially weaker . additional removal of the poorly conserved n - terminal region did not detectably compromise interactions ( table s1 ) . although it has previously been shown that ser487 and ser491 are both phosphorylated by ck2 in  vivo , ser487 is not conserved in tel2 orthologs ( figure  1c ) , suggesting that ck2 phosphorylation of ser491 constitutes the switch for tel2 recognition by  pih1d1 and its orthologs in all eukaryotes . furthermore , sequence comparison reveals that ser491 in human tel2 is embedded in an absolutely conserved dpsdd motif ( figure  1c ) , and , accordingly , analysis showed that pih1d1 interacts only with tel2 phosphopeptides in which the dpsdd motif is intact ( figure  1d ) . in order to further probe interactions with the conserved acidic ck2 substrate motif within tel2 , and based on our structural studies described below , all subsequent itc titrations used shorter ( 8-mer ) peptides ( table s1 ) . in these experiments , phosphopeptides in which each of the three aspartate residues were individually substituted by alanine all failed to show any significant quantifiable binding , and replacement of pser491 with glu as a phosphopeptide mimic also severely compromised interactions ( figure  1e ) . indeed , substitution of pser491 with pthr significantly reduced binding , even in the context of longer tel2 peptides , revealing a marked discrimination in favor of phosphoserine - containing motifs ( figure  1f ) . altogether , these data show that the pih1 homology region encompasses two structurally and functionally separable domains  hereafter referred to as pih - n and pih - c  which are linked by an intervening region of variable length and sequence ( figure  s1 ) . furthermore , they suggest that the major interacting site for the pih - n domain of pih1d1 ( and budding yeast pih1 ) is a  short dpsdd motif that encompasses the only amino acids conserved between tel2 orthologs from humans to yeast . to investigate the molecular basis of the phosphopeptide - specific binding activity of the pih - n domain , we solved the crystal structures of pih1d1 domain fragments alone and bound to the tel2 pser491 peptide ( table s2 ) . the structure of the peptide - free pih1d1 51180 fragment shows that the pih - n domain adopts an unusual  topology ( figure  2a ) that is unique among previously reported phosphopeptide - interacting proteins  and modules . the structure of the pih - n domain in the pih1d1-ptel2 phosphopeptide complex ( figure  2b ) is essentially identical except for the fact that helix 1 located between 2 and 3 is disordered . in the structure of the pih1d1-ptel2 complex , 11 residues of the peptide are defined in the electron density maps and bind to a shallow , positively charged groove formed by the  sheet and an extended c - terminal segment ( figures 2b and s2 ) . five n - terminal residues , including pser487 , are disordered , suggesting that the small binding contribution of the additional phosphosite results from weak , nonspecific electrostatic interactions that are not associated with a single defined conformation . the most significant interactions of pih1d1 are formed with the core dpsdd motif in tel2 , to the extent that the only intermolecular hydrogen bonds seen in the  complex involve these four residues ( figure  2c ) . indeed , the  structure nicely explains the high degree of conservation of  the dpsdd motif . although it appears that substitution of aspartate in the pser 1 position with glutamate could be accommodated , the pattern of hydrogen bonding of the aspartates in the  + 1 and  + 2 positions along with their steric environment indicates that even conservative substitution with glutamate would be detrimental to binding ( figure  2d ) . from the perspective of the pih - n domain itself , only three ( lys57 , lys64 , and arg168 ) of the five basic residues that contribute to the positively charged binding surface make hydrogen bonding interactions with tel2 . of these , only lys57 and lys64 interact directly with the tel2 pser491 phosphoryl group and constitute the only basic residues conserved across pih1d1 orthologs from yeast to humans ( figure  s1b ) . arg168 forms the base of the phosphopeptide binding site , making hydrogen  bonds with main - chain atoms of the bound ligand in a manner  reminiscent of the binding mode observed in brca1 c  terminus phosphopeptide complexes ( brca1 arg1699 and mdc1 arg1933 ) ( clapperton et  al . , 2004 , stucki et  al . , accordingly , mutation of each of these residues to alanine ( k57a , k64a , and r168a ) essentially abolished tel2 phosphopeptide binding as judged by itc , and smaller but nonetheless significant effects were observed for mutations of basic residues at more peripheral positions ( figure  2e ; table s1 ) . in addition , both k57a and k64a mutants of full - length pih1d1 failed to immunoprecipitate endogenous tel2 from whole - cell extract but nonetheless retained binding to the other components of  the r2tp complex mediated through the pih - c region ( figure  2f ) . interestingly , both phosphopeptide - interacting lysines are absent in pih1d2 but are substituted with arginine in another pih1d1 ortholog , kintoun , a regulator of dynein assembly ( omran et  al . , 2008 ) . although we have been unable to test pih1d2-ptel2 interactions by itc , the lack of conservation at  these crucial positions renders a phosphopeptide binding activity unlikely . however , the pih - n domain of kintoun does show some weak , but nonetheless significant , binding to the ptel2 peptide ( figure  s3 ) , suggestive of a phosphopeptide binding capacity that is , presumably , associated with an altered overall target sequence specificity . our data raised the intriguing possibility that r2tp complex substrates may be defined by their ability to bind to pih1d1 in a phosphopeptide - specific manner . to investigate this further and identify previously unreported r2tp complex substrates , we conducted a comparative proteomic analysis of wild - type ( wt ) pih1d1 and the k64a mutant , which is compromised for  phosphopeptide binding ( table s3 ) . among others , tel2 , snrp116 , ubr5 , and rna polymerase ii subunit rpb1 were identified by mass  spectrometry specifically with wt pih1d1 , but not the k64a mutant , and were confirmed by immunoprecipitation and western blotting ( figure  3a ) . immunoaffinity purified snrp116 and ubr5 also bound to wt pih1d1 fused to glutathionine s - transferase ( gst ) but not gst - k64a pih1d1 . furthermore , lambda phosphatase treatment of the immunoprecipitated snrp116 and ubr5 disrupted their binding to gst - wt pih1d1 , indicating that the interactions are phosphorylation dependent , whereas rpap3 bound to the  gst - tagged pih1d1 regardless of phosphorylation status or the integrity of lys64 ( figure  3b ) . these results reveal that , analogous to tel2 binding , snrp116 and ubr5 interact with the pih domain of pih1d1 in a phosphorylation - dependent manner . next , we determined a minimal consensus sequence necessary for tel2 phosphopeptide interaction with the pih - n domain ( figure  4a ) . to this end , we assayed binding to a tel2 pep - spot substitution array in which each residue in the polypeptide was substituted by all other amino acids . from this analysis , we derived a phosphopeptide binding consensus motif ( d[s / t]dd[d / e ] ) that was then used in an in  silico screen to identify additional potential substrates of the r2tp complex . proteins containing the pih - n domain binding consensus sequence included ecd , ubr5 , ptges3 , eif5b , trp12 , and sfrs18 ( figure  4b ) . of these , we chose to further characterize and validate the interaction with ecd , which is involved in p53 stabilization and regulation of retinoblastoma phosphorylation ( kim et  al . , 2009 , zhang et  al . , 2006 ) and contains two pih - n domain binding sequences surrounding ck2 consensus phosphorylation sites ser505 and ser518 . in contrast to the unphosphorylated ecd peptide , an ecd peptide phosphorylated at ser505 efficiently pulled down pih1d1 from whole - cell extract ( figure  4c ) and exhibited a binding affinity of 16  m in itc analysis ( figure  4d ) . ecd also immunoprecipitated wt pih1d1 but failed to interact with pih1d1 k64a ( figure  4e ) . conversely , mutation of the predicted ck2 sites in ecd ( ser505 and ser518 ) abolished interaction with pih1d1 ( figure  4f ) . thus , like tel2 , ecd is recruited to the r2tp complex through interactions of the pih - n domain of pih1d1 with the conserved dsdd motif , possibly in a ck2 phosphorylation - dependent manner . given that ecd , ubr5 , and tel2 are important for the regulation of p53 ( ling and lin , 2011 , reid et  al . , 2013 , smits , 2012 , takai et  al . , 2007 ) , we sought to determine if pih1d1 also impacts on p53 function . to this end , we depleted pih1d1 and analyzed p53 stability and phosphorylation on ser15 after dna damage . depletion of pih1d1 with two different  small interfering rnas ( sirnas ) resulted in reduced p53 levels and a loss of ser15 phosphorylation after dna damage ( figures s4a and s4b ) . to  determine whether this effect is dependent on pih - n phosphopeptide binding activity , we stably transfected retinal pigment epithelium cells with wt pih1d1 , pih1d1 k64a mutant , or empty vector and selected single clones expressing pih1d1 at levels similar to endogenous ( figure  s4c ) . depletion of pih1d1 with sirna targeting the 5 untranslated region of pih1d1 from cells reconstituted with wt pih1d1 rescued levels of p53 protein and phosphorylation after dna damage to normal levels , which was in contrast to pih1d1 k64a . therefore , we conclude that the phosphopeptide binding ability of pih1d1 is crucial for the function of r2tp complex ( figure  4 g ) . along with hsp90 , the r2tp complex has been implicated in the  assembly of large protein or ribonucleoprotein complexes in the  cytoplasm and the nucleus . in contrast to the two aaa+  atpases that are present in many different multisubunit complexes ( ruvbl1 and ruvbl2 ) , pih1d1 and rpap3 are specific to the r2tp complex and are likely to define substrate binding , potentially via interaction with the tetratricopeptide repeats of rpap3 . nevertheless , ruvbl1 and ruvbl2 are known to be important for assembly of r2tp complex substrates into an appropriate functional conformation ( machado - pinilla et  al . , 2012 ) . however , a mechanistic explanation that defines how the r2tp complex is able to recognize and facilitate assembly of diverse substrates is largely unknown . the results presented here reveal a previously unrecognized phosphopeptide binding domain ( pih - n ) within the pih1d1 subunit of the r2tp complex that preferentially binds to acidic phosphorylated proteins with high specificity . structural analysis of the pih - n domain bound to the tel2 phosphopeptide established a unique mechanism of phosphopeptide recognition and identified lys57 and lys64 in pih1d1 as essential for phosphopeptide binding . both lysines , as well as the ck2 phosphorylated tel2 binding motif dpsdd , are highly conserved in pih1d1 and tel2 orthologs from yeasts to human , indicating a common mechanism for substrate recognition by pih - n domain . through proteomic and in  silico analysis , we demonstrate that the pih - n domain binds to snrp116 , ubr5 , rpb1 , and ecd in a phosphorylation - dependent manner analogous to tel2 . thus , we suggest that substrates are marked for assembly by the r2tp complex through phosphorylation of a short , highly acidic motif by ck2 ( or other acidophilic kinases ) , which is then recognized by the phosphopeptide binding function of the pih1d1 subunit . the emergence of hsp90 inhibitors as successful cancer therapeutics has highlighted the importance of protein complex assembly and chaperone activities in cancer cells . given that the r2tp complex works in conjunction with hsp90 to assemble multisubunit protein and ribonucleoprotein complexes , r2tp complex inhibitors could also be of value in a clinical setting . small molecules that block the pih - n phosphopeptide binding domain by mimicking the dpsdd motif would be predicted to prevent the rt2p complex from recognizing its substrates . notably , the pih1d1 subunit is overexpressed in several breast cancer cell lines ( kamano et  al . , 2013 ) , which may reflect a dependency of the tumor growth for r2tp complex activity . therefore , it is possible that inhibitors of pih - n domain interactions could be used as an anticancer therapeutic for tumors that are overreliant on such chaperone activities . recombinant selenomethionine - substituted human pih1d1 ( 51180 ) was crystallized in complex with a diphosphopeptide ( nh2-yagsdpsdldpsddefvpy - conh ) encompassing residues 483497 of human tel2 by sitting drop vapor diffusion with an impax nano - dispensing robot . crystals grew from a 8  mg / ml solution of complex in 20  mm tris ( ph 8.0 ) , 150  mm nacl , and 0.5  mm tris(2-carboxyethyl)phosphine ( tcep ) equilibrated well solution containing 32% w / v polyethylene glycol ( peg ) 4000 , 100  mm sodium acetate , and 100  mm tris ( ph 8.5 ) as precipitant , transferred to a cryo - protectant containing protein buffer  and mother liquor supplemented with 20% v / v glycerol , and flash frozen in liquid nitrogen . images were indexed , integrated , and scaled with mosflm / scala ( ccp4 ) . phases for the pih1d1-ptel2 complex were determined by single - wavelength anomalous diffraction and were of sufficient quality to enable a partial model of the two pih1d1-ptel2 complexes in the crystallographic asymmetric unit to be built automatically with buccaneer ( ccp4 ) . all manual model building was carried out with coot ( emsley and cowtan , 2004 ) , and structure refinement was carried out with phenix ( terwilliger , 2002 ) . the structure of unbound pih1d1 ( 51180 ) was crystallized by microseeding and vapor diffusion from 2% peg 400 ( v / v ) , 20% methoxy peg 5000 , and 0.1  m imidazole ( ph 7.0 ) . the structure was determined by molecular replacement using the final refined protein coordinates derived from the structure of the complex as a search model with phaser ( mccoy , 2007 ) and refined against data extending to a 1.58   resolution . proteins were prepared by dialysis against 20  mm tris ( ph 8.0 ) , 150  mm nacl , and 0.5  mm tcep . synthetic phosphopeptides ( cambridge peptides ) were desalted with nap5 columns ( ge healthcare ) equilibrated in 20  mm tris ( ph 8.0 ) , 150  mm nacl , and 0.5  mm tcep and diluted to the appropriate concentration with dialysis buffer . protein concentrations were determined by uv spectrometry . a typical experiment involved 19   2  l injections of 800  m peptide from the injection syringe into 80  m protein in the sample cell . sypro ruby - stained polyacrylamide gel slices ( 12  mm ) were excised with a scalpel and processed for mass spectrometry with the janus automated liquid handling system ( perkinelmer ) . / t]dd[e / d ] were identified with the prosite database ( http://prosite.expasy.org ) tools . the resulting list of candidates was filtered with the criteria that the site is at least conserved in mammals and that it has previously been reported as phosphorylated in  vivo . for peptide pull - down assay , a detailed summary of all procedures used in this study can be found in the supplemental experimental procedures .
summarythe r2tp cochaperone complex plays a critical role in the assembly of multisubunit machines , including small nucleolar ribonucleoproteins ( snornps ) , rna polymerase ii , and the mtorc1 and smg1 kinase complexes , but the molecular basis of substrate recognition remains unclear . here , we describe a phosphopeptide binding domain ( pih - n ) in the pih1d1 subunit of the r2tp complex that preferentially binds to highly acidic phosphorylated proteins . a cocrystal structure of a pih - n domain / tel2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which lys57 and 64 in pih1d1 , along with a conserved dpsdd phosphopeptide motif within tel2 , are essential and sufficient for binding . 
 proteomic analysis of pih1d1 interactors identified r2tp complex substrates that are recruited by the pih - n domain in a sequence - specific and phosphorylation - dependent manner suggestive of a common mechanism of substrate recognition . 
 we propose that protein complexes assembled by the r2tp complex are defined by phosphorylation of a specific motif and recognition by the pih1d1 subunit .