Protein functional epitopes: hot spots, dynamics and combinatorial libraries

doi:10.1016/S0959-440X(00)00216-5

Current Opinion in Structural Biology

Volume 11, Issue 3, 1 June 2001, Pages 364-369

https://doi.org/10.1016/S0959-440X(00)00216-5 Get rights and content

Abstract

Recent studies increasingly point to the importance of structural flexibility and plasticity in proteins, highlighting the evolutionary advantage. There are an increasing number of cases in which given, presumably specific, binding sites have been shown to bind a range of ligands with different compositions and shapes. These studies have also revealed that evolution tends to find convergent solutions for stable intermolecular associations, largely via conservation of polar residues as hot spots of binding energy. On the other hand, the ability to bind multiple ligands at a given site is largely derived from hinge-based motions. The consideration of these two factors in functional epitopes allows more realism and robustness in the description of protein binding surfaces and, as such, in applications to mutants, modeled structures and design. Efficient multiple structure comparison and hinge-bending structure comparison tools enable the construction of combinatorial binding epitope libraries.

Introduction

Considerable effort has been invested over the past few years in schemes designed to identify functional epitopes on protein surfaces. Reliable prediction of functional epitopes has immediate implications for drug/inhibitor design and for protein engineering. In many cases, attempts to detect binding sites through geometrical or chemical characteristics have failed, however, to provide clear-cut leads.

Protein binding sites have neither the largest total buried surface area nor the most extensive nonpolar buried surface area. They cannot be uniquely distinguished by their electrostatic characteristics, as observed by schemes such as unsatisfied buried charges, or the number of hydrogen bonds [1]. Although the geometry of molecular surfaces has provided clues to binding sites on enzyme surfaces, which are often shaped as the largest or deepest clefts on the surface 2., 3., none were found for protein–protein binding sites.

Furthermore, recent experiments have clearly illustrated that even presumably specific binding sites may still bind a range of ligands with different compositions and shapes 4••., 5•.. These experiments point to the contribution of frequently conserved residues to stabilizing the association and molecular flexibility, which is largely the outcome of hinge-based motions, allowing expansion or shrinking of the binding pockets. Plasticity implies a range of conformational substates, with low energy barriers. Therefore, the distribution of the conformational substrates may be easily changed. Different receptor conformers may bind different ligands, with the population of the conformational substrates redistributed, sustaining the equilibrium, and further driving intermolecular association [6••].

In this review, we focus on two properties of functional epitopes, conserved energetic hot spots and molecular flexibility. The advantage of these compared to a detailed description of molecular surfaces is that they provide robustness, allow application to modeled structures and implicitly take account of mutational events. Efficient computational tools enable the use of these properties to construct combinatorial libraries of functional epitopes, allowing genomic applications.

Section snippets

Functional epitopes: dynamics and conservation

In a thought-provoking comment, Van Regenmortel [7••] argued that analyzing the interactions between biological molecules can not be reduced to the description of (static) molecular structures. Integrated functional approaches need to consider the binding partner and the time component of the interaction. The function of a protein and its properties are decided not only by the static folded three-dimensional structure, but also by the distribution and redistributions of the populations of its

Protein conformational flexibility

Conformational flexibility is largely expressed through the range of substates around the native state and their (low) barrier heights. The role of conformational flexibility in binding and regulation through conformer selection has been extensively discussed in a recent series of review articles ([6••] and references therein). Nature's way of making use of protein flexibility to expand the range and type of ligands [12] has been recreated in the laboratory in two experiments by employing the

Conserved hot spots are largely polar residues

Interface residues contributing the most to the binding free energy (more than 2 kcal/mol) are known as hot spots 20., 21., 22•.. Experimentally, hot spots may be located via traditional or shotgun alanine mutations, through identifying large binding free energy change from the mutation. [23•]. A computational alanine-scanning method combining explicit molecular mechanical energies and a continuum solvation model for calculating protein–peptide interaction free energies has qualitatively

Binding partners, time events and convergent evolution

In considering binding partners, dynamics and conservation of functional epitopes, there are a number of possible scenarios. In the first, a functional protein binds multiple ligands simultaneously. A multienzyme complex may tolerate hardly any amino acid substitutions [27]. The glycolytic pathway enzymes that aggregate in the glycosomes of trypanosomes and Leishmania constitute an example of such a case, limiting adaptability to altered multiple simultaneous ligand binding.

In the second, more

Combinatorial epitope libraries: efficient computational tools

To conclude, functional epitopes frequently consist of a set of groups of polar residues rotated with respect to each other, with a range of hinge-based motions. These hinges may be distributed over a number of backbone angles and their extent is a function of the conformer selection, depending on the incoming ligands. The polar residues may interact with the ligand directly and/or indirectly through a network of conserved and nonconserved inter-residue communications.

For the purpose of drug

Conclusions

Traditional attempts to detect binding sites through geometrical or chemical characteristics often fail to provide clear-cut solutions. Here we highlight two frequently observed properties of binding sites: flexibility and the presence of polar residue hot spots. The topology of the protein dictates its more (and less) dynamic regions. Flexibility implicates an ensemble of conformers. Hence, binding sites are not static entities and should not be considered as such. Rather, they are defined by

Acknowledgements

We thank JV Maizel for discussions and for encouragement. We thank our students Tal Elkayam and Maxim Shatsky for generating Fig. 1 and Fig. 2, respectively. M Shatsky has written the program used for creating the alignment in Fig. 2. The research of R Nussinov and HJ Wolfson in Israel has been supported in part by the Magnet grant, by the Ministry of Science grant, and by the ‘Center of Excellence in Geometric Computing and its Applications’ funded by the Israel Science Foundation

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest
•• of outstanding interest

References (38)

K.P Peters et al.
The automatic search for ligand binding sites in proteins of known three-dimensional structure using only geometric criteria
J Mol Biol
(1996)
D Tondi et al.
Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase
Chem Biol
(1999)
O Keskin et al.
Proteins with similar architecture exhibit similar large-scale dynamic behavior
Biophys J
(2000)
J.K Kranz et al.
RNA recognition by human U1A protein is mediated by a network of local cooperative interactions that create the optimal binding surface
J Mol Biol
(1999)
E.J Sundberg et al.
Luxury accommodations: the expanding role of structural plasticity in protein-protein interaction
Structure
(2000)
S Chakravarty et al.
Residue depth: a novel parameter for the analysis of protein structure and stability
Structure
(1999)
P.E Wright et al.
Intrinsically unstructured proteins: reassessing the protein structure-function paradigm
J Mol Biol
(1999)
A.A Bogan et al.
Anatomy of hot spots in protein interfaces
J Mol Biol
(1998)
C.J Tsai et al.
A dataset of protein–protein interfaces generated with a sequence-order-independent comparison technique
J Mol Biol
(1996)
B Kisters-Woike et al.
On the conservation of protein sequences in evolution
Trends Biochem Sci
(2000)

C.S Goh et al.

Co-evolution of proteins with their interaction partners

J Mol Biol

(2000)

S.A Gillmor et al.

Compromise and accommodation in ecotin, a dimeric macromolecular inhibitor of serine proteases

J Mol Biol

(2000)

P Koehl et al.

De novo protein design. II plasticity in sequence space

J Mol Biol

(1999)

F.C Bernstein et al.

The protein databank: a computer-based archival file for macromolecular structures

J Mol Biol

(1977)

R Norel et al.

Examination of shape complementarity in docking of unbound proteins

Proteins

(1999)

R.A Laskowski et al.

Protein clefts in molecular recognition and function

Protein Sci

(1996)

W.L DeLano et al.

Convergent solutions to binding at a protein–protein interface

Science

(2000)

S Kumar et al.

Folding and binding cascadesdynamic landscapes and population shifts

Protein Sci

(2000)

V.an Regenmortel

MHV: Molecular recognition in the post-reductionist era

J Mol Recog

(1999)

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ReviewProtein functional epitopes: hot spots, dynamics and combinatorial libraries

Abstract

Introduction

Section snippets

Functional epitopes: dynamics and conservation

Protein conformational flexibility

Conserved hot spots are largely polar residues

Binding partners, time events and convergent evolution

Combinatorial epitope libraries: efficient computational tools

Conclusions

Acknowledgements

References and recommended reading

J Mol Biol

Chem Biol

Biophys J

J Mol Biol

Structure

Structure

J Mol Biol

J Mol Biol

J Mol Biol

Trends Biochem Sci

J Mol Biol

J Mol Biol

J Mol Biol

J Mol Biol

Examination of shape complementarity in docking of unbound proteins

Proteins

Protein clefts in molecular recognition and function

Protein Sci

Convergent solutions to binding at a protein–protein interface

Science

Folding and binding cascadesdynamic landscapes and population shifts

Protein Sci

MHV: Molecular recognition in the post-reductionist era

J Mol Recog

Review
Protein functional epitopes: hot spots, dynamics and combinatorial libraries