Identification of Hydrophobic Interfaces in Protein-Ligand Complexes by Selective Saturation Transfer NMR Spectroscopy.

Fabien Ferrage,David Cowburn,Kaushik Dutta

doi:10.3390/molecules201219824

Abstract

The proper characterization of protein-ligand interfaces is essential for structural biology, with implications ranging from the fundamental understanding of biological processes to pharmacology. Nuclear magnetic resonance is a powerful technique for such studies. We propose a novel approach to the direct determination of the likely pose of a peptide ligand onto a protein partner, by using frequency-selective cross-saturation with a low stringency isotopic labeling methods. Our method illustrates a complex of the Src homology 3 domain of C-terminal Src kinase with a peptide from the proline-enriched tyrosine phosphatase.

Highlights

The accurate characterization of protein-protein interfaces is a key element for the understanding of biological mechanisms at a sub molecular level
The most used nuclear magnetic resonance (NMR) method consists in following the chemical shift perturbations on one protein upon titration of the binding partner
This experiment is very sensitive but it provides ambiguous and possibly inaccurate data. This information can be used to define the interface and dock the structure of a complex [5]. Deficits of this approach include the indirect nature of chemical shift perturbation on complex formation, and ad hoc knowledge-based interpretation

Summary

Introduction

The accurate characterization of protein-protein interfaces is a key element for the understanding of biological mechanisms at a sub molecular level. In contrast cross-saturation [7,8,9], REDuced/Standard Proton density INTerface identification (REDSPRINT) [10] and the use of enhanced relaxation at the interface [11,12] can provide accurate but partly ambiguous information about the complex. Among all these methods, cross-saturation has the advantage of being both accurate and applicable to large complexes. Hspereec,ttrhails raelgloiwonedwuisthtosicganrriyficant cross-osnatuserlaetcitoivneosnataulripathioanticoof faarpormotaotniactseiddeb-icnhdainings poafrtthnerRiEnDtPhRe Oα lapbroetloend psproectetrianl. rTehgeioenxpweirthiment was dseigmniofincsatnrtacterodsso-nsatuhreatcionmopnleaxlipohf aStricc ohfoamroomloagtiyc s3iddeo-cmhaaiinns (oSfHth3e)RoEfDCP-RteOrmlaibnealeldSrpcroKteinina.sTeh(eCSK) with aexrpeedruimceedntpwroatsodnedmeonnssittryat[e1d7]oanntdhethceom25prleexsiodfuSerclohnogmpoelpogtiyd3e fdroommatinhe(SpHro3)lionfe-Ce-nterrimchiendaltSyrrcosine phospKhinaatasese(C(PSKEP) w) [i1th9]a. reduced proton density [17] and the 25 residue long peptide from the prolineenriched tyrosine phosphatase (PEP) [19]

Results and Discussion

Experimental Section

Conclusions