Dean R. Artis

Comprehensive and Quantitative Mapping of Energy Landscapes for Protein-Protein Interactions by Rapid Combinatorial Scanning

A novel, quantitative saturation (QS) scanning strategy was developed to obtain a comprehensive data base of the structural and functional effects of all possible mutations across a large protein-protein interface. The QS scan approach was applied to the high affinity site of human growth hormone (hGH) for binding to its receptor (hGHR). Although the published structure-function data base describing this system is probably the most extensive for any large protein-protein interface, it is nonetheless too sparse to accurately describe the nature of the energetics governing the interaction. Our comprehensive data base affords a complete view of the binding site and provides important new insights into the general principles underlying protein-protein interactions. The hGH binding interface is highly adaptable to mutations, but the nature of the tolerated mutations challenges generally accepted views about the evolutionary and biophysical pressures governing protein-protein interactions. Many substitutions that would be considered chemically conservative are not tolerated, while conversely, many non-conservative substitutions can be accommodated. Furthermore, conservation across species is a poor predictor of the chemical character of tolerated substitutions across the interface. Numerous deviations from generally accepted expectations indicate that mutational tolerance is highly context dependent and, furthermore, cannot be predicted by our current knowledge base. The type of data produced by the comprehensive QS scan can fill the gaps in the structure-function matrix. The compilation of analogous data bases from studies of other protein-protein interactions should greatly aid the development of computational methods for explaining and designing molecular recognition.

Chapter 28. Structure-Based Design from Flexible Ligands

Publisher Summary This chapter discusses some strategies that have been used to obtain structural information from the well flexible molecules. While these strategies may not yield a model that has the atomic level of detail afforded by X-ray crystallography, examples illustrates that even approximate structural models can provide a useful springboard for the discovery of novel lead series. Although the chapter discusses the peptides, it is clear that similar strategies can be applied to other compounds that can be assembled in a modular fashion, including hits obtained from combinatorial synthesis. It is clear from the examples mentioned in the chapter that many powerful tools exist for deducing putative bioactive conformations of flexible molecules. The biological importance of the Phe-Trp-Lys-Thr turn in somatostatin is underscored by the many potent, structured peptide analogs that have been developed. Successive rigidification of the arginine-glycine-aspartic acid (RGD) epitope in cyclic peptides has led in two cases to models that directed the design of novel nonpeptide lead compounds. Angiotensin II and bradykinin illustrate how local conformational constraints can be combined with cyclization to the sequentially develop high-confidence structural models. Excepting the GPIIb/IIIa antagonists, however, few compelling examples exist, in which these types of models have been used successfully to design fundamentally the new chemical entities.

Receptor-binding conformation of the “ELR” motif of IL-8: X-ray structure of the L5C/H33C variant at 2.35 Å resolution

The “ELR” (Glu‐Leu‐Arg) tripeptide sequence near the N‐terminus of interleukin‐8 (IL‐8) contributes a large part of the receptor binding free energy. Prior X‐ray and nuclear magnetic resonance (NMR) structures of IL‐8 have shown this region of the molecule to be highly mobile. We reasoned that a hydrophobic interaction between the leucine and the neighboring β‐turn might exist in the receptor binding conformation of the N‐terminus. To test this hypothesis, we mutated two residues to cysteine and connected the N‐terminus to the β‐turn. The mutant retains receptor binding affinity reasonably close to wild type and allows the characterization of a high‐affinity conformation that may be useful in the design of small IL‐8 mimics. The L5C/H33C mutant is refined to R‐values of R = 20.6% and Rfree = 27.7% at 2.35 Å resolution. Other receptor binding determinants reside in the “N‐loop” found after “ELR” and preceding the first β‐strand. All available structures of IL‐8 have been found with one of two distinct N‐loop conformations. One of these is relevant for receptor binding, based on NMR results with receptor peptides. The other conformation obscures the receptor‐peptide binding surface and may have an undetermined but necessarily different function. Proteins 2000;38:361–367.

Transfer of a protein binding epitope to a minimal designed peptide.

Results from protein mutagenesis and x‐ray crystallographic studies of the multidomain protein Vascular Cell Adhesion Molecule (VCAM) were used to design cyclic octapeptides that retain the critical structural and binding elements of the epitope of VCAM in the interaction with the integrin α4β1 (VLA‐4). Changes in the activities of peptide analogues correlated with the relative activities of protein mutants of VCAM, and predicted the properties of two new mutants that bound α4β1 with improved affinity vs wild type protein. The nmr structures of two peptides revealed a high degree of similarity to the structure of the VCAM binding epitope. These results demonstrate that a compact binding epitope identified via protein structure–function studies may be transferred to a synthetically accessible small peptide with the key structure–activity relationships intact.

STRUCTURAL CHANGE AND RECEPTOR BINDING IN A CHEMOKINE MUTANT WITH A REARRANGED DISULFIDE : X-RAY STRUCTURE OF E38C/C50A IL-8 AT 2 A RESOLUTION

The characteristic CXC chemokine disulfide core of interleukin‐8 (IL‐8) has been rearranged in a variant replacing the 9—50 disulfide with a 9—38 disulfide. The new variant has been characterized by its binding affinity to IL‐8 receptors A and B and the erythrocyte receptor DARC. This variant binds the three receptors with affinities between 500‐ and 2,500‐fold lower than wild‐type IL‐8. Binding affinity results are also reported for the variant with alanine substituted for both cysteines 9 and 50. The Glu38 → Cys/Cys50 → Ala IL‐8 crystallizes in space group P212121 with cell parameters a = 46.4, b = 49.2, and c = 69.5 Å, and has been refined to an R‐value of 19.4% for data from 10 to 2 Å resolution. Analysis of the structure confirms the new disulfide arrangement and suggests that changes at Ile10 may be the principal cause of the lowered affinities.