Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope

Pires, Juliana Rico; Taha-Nejad, F.; Toepert, Florian; Ast, Thomas; Hoffmüller, Ulrich; Schneider‐Mergener, Jens; Kühne, Ronald; Macias, María J.; Oschkinat, Hartmut

doi:10.1006/jmbi.2000.5199

Cited by 109 publications

(155 citation statements)

References 26 publications

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“…ε pro abx Þ. However, the mean epistasis score was also unable to separate all three known stabilizing hYAP65 WW domain mutations present in our dataset from the bulk of the mutations as well as two known activityenhancing mutations (19)(20)(21)(22) (Fig. 2C).…”

Section: Resultsmentioning

confidence: 79%

“…Because stabilizing mutations could potentially rescue many destabilizing mutations, the simplest strategy to find them would rely on the expectation that stabilizing mutations are among the most highly represented mutations after selection. As a gold standard, we used three known stabilizing hYAP65 WW domain mutations (19,20) present in the dataset (A20R, L30K, and D34T), which under this expectation, should become highly enriched. However, for these three mutations, postselection representation was not a useful predictor of stability (Fig.…”

Section: Resultsmentioning

confidence: 99%

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A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

Araya¹,

Fowler

Chen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

240

264

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The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein's structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein's function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered.deep mutational scanning | epistasis | high-throughput DNA sequencing

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Section: Resultsmentioning

confidence: 79%

Section: Resultsmentioning

confidence: 99%

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

Araya¹,

Fowler

Chen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

240

264

View full text Add to dashboard Cite

show abstract

“…Interestingly, the wild-type sequence of the YAP and TAZ WW2 domains are unique for having these negatively charged residues. The weak affinity of the YAP WW1 domain for PY motifs (Macias et al 1996;Pires et al 2001;Toepert et al 2001) and the presence of a negatively charged patch in the YAP WW2 domain that prevents binding to a pT[PY] motif explain our previous result that YAP does not interact efficiently with linker phosphorylated Smad3 (Supplemental Fig. 5F-H; Alarcon et al 2009).…”

Section: Basis For Nedd4l and Pin1 Recognition Of The Smad3 Linker Phmentioning

confidence: 83%

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

Aragón

Goerner²,

Zaromytidou³

et al. 2011

Genes Dev.

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217

286

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When directed to the nucleus by TGF-b or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-b pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-b signal delivery to turnover of the Smad signal transducers.

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“…Little is currently known about the functional importance of the interdomain linker in proteins with multiple WW domains. Although a number of structures of individual WW domains have been solved (17)(18)(19)(20)(21)(22), only two structures of tandem WW domains, namely those of yeast Prp40 and Drosophila Su(dx), have been determined, and their structures are markedly different (7,14). So far, the relationship between the functional diversity and the structural organization of multiple WW domains remains poorly characterized.…”

mentioning

confidence: 99%

Structure and Function of the Two Tandem WW Domains of the Pre-mRNA Splicing Factor FBP21 (Formin-binding Protein 21)

Huang

Beullens

Zhang

et al. 2009

Journal of Biological Chemistry

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Human FBP21 (formin-binding protein 21) contains a matrin-type zinc finger and two tandem WW domains. It is a component of the spliceosomes and interacts with several established splicing factors. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and that its splicing activation function and interaction with the splicing factor SIPP1 (splicing factor that interacts with PQBP1 and PP1) are both mediated by the two tandem WW domains of group III. We determined the solution structure of the tandem WW domains of FBP21 and found that the WW domains recognize peptide ligands containing either group II (PPLP) or group III (PPR) motifs. The binding interfaces involve both the XP and XP2 grooves of the two WW domains. Significantly, the tandem WW domains of FBP21 are connected by a highly flexible region, enabling their simultaneous interaction with two proline-rich motifs of SIPP1. The strong interaction between SIPP1 and FBP21 can be explained by the conjugation of two low affinity interactions with the tandem WW domains. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains.Gene expression in eukaryotic cells involves several steps, including transcription, mRNA processing, and export. Pre-mRNA splicing takes place in the spliceosome, a highly dynamic ribonucleoprotein particle that consists of five small nuclear RNAs and at least 150 proteins. Small nuclear ribonucleoproteins (snRNPs) 3 and numerous protein factors are essential for the formation of the active spliceosome (1, 2). In budding yeast, the splicing factor Prp40 participates in crossintron bridging by interacting with the branch point-binding protein (BBP) and the U5 snRNP component Prp8. Prp40 contacts the 5Ј splice site and interacts with BBP, bringing the 5Ј splice site and the branch point in spatial proximity. These interactions are believed to be conserved in mammals (3-5). FBP21 (formin-binding protein 21), the mammalian Prp40-like protein, colocalizes with splicing factors in nuclear storage sites for pre-mRNA splicing factors. In addition, FBP21 is a component of the mammalian spliceosomal A/B complex and is associated with U2 snRNPs (6). FBP21 interacts directly with the splicing factors U1 snRNP protein U1C, the core snRNP proteins SmB and SmBЈ, and the branch point-binding protein SF1/mBBP, suggesting that it may also play a role in crossintron bridging of U1 and U2 snRNPs in the spliceosomes. FBP21 contains a matrin-type zinc finger and two group III WW domains ( Fig. 1) that are structurally related to those of the established splicing factors U1C and Prp40, respectively (6, 7). The binding of FBP21 to splicing factors is mediated by its tandem WW domains, which represent interaction modules for proline-rich ligands (4, 8, 9). Although the above data strongly suggest that FBP21 has a role in pre-mRNA splicing, there are no in vivo data to support this contention. The splicing...

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Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope

Cited by 109 publications

References 26 publications

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

Structure and Function of the Two Tandem WW Domains of the Pre-mRNA Splicing Factor FBP21 (Formin-binding Protein 21)

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