On the evolutionary conservation of hydrogen bonds made by buried polar amino acids: the hidden joists, braces and trusses of protein architecture.

BACKGROUND: The hydrogen bond patterns between mainchain atoms in protein structures not only give rise to regular secondary structures but also satisfy mainchain hydrogen bond potential. However, not all mainchain atoms can be satisfied through hydrogen bond interactions that arise in regular secondary structures; in some locations sidechain-to-mainchain hydrogen bonds are required to provide polar group satisfaction. Buried polar residues that are hydrogen-bonded to mainchain amide atoms tend to be highly conserved within protein families, confirming that mainchain architecture is a critical restraint on the evolution of proteins. We have investigated the stabilizing roles of buried polar sidechains on the backbones of protein structures by performing an analysis of solvent inaccessible residues that are entirely conserved within protein families and superfamilies and hydrogen bonded to an equivalent mainchain atom in each family member. RESULTS: We show that polar and sometimes charged sidechains form hydrogen bonds to mainchain atoms in the cores of proteins in a manner that has been conserved in evolution. Although particular motifs have previously been identified where buried polar residues have conserved roles in stabilizing protein structure, for example in helix capping, we demonstrate that such interactions occur in a range of architectures and highlight those polar amino acid types that fulfil these roles. We show that these buried polar residues often span elements of secondary structure and provide stabilizing interactions of the overall protein architecture. CONCLUSIONS: Conservation of buried polar residues and the hydrogen-bond interactions that they form implies an important role for maintaining protein structure, contributing strong restraints on amino acid substitutions during divergent protein evolution. Our analysis sheds light on the important stabilizing roles of these residues in protein architecture and provides further insight into factors influencing the evolution of protein families and superfamilies.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Structural Biology and Drug Discovery of Difficult Targets: The Limits of Ligandability

Over the past decade, researchers in the pharmaceutical industry and academia have made retrospective analyses of successful drug campaigns in order to establish “rules” to guide the selection of new target proteins. They have identified features that are considered undesirable and some that make targets “unligandable.” This review focuses on the factors that make targets difficult: featureless binding sites, the lack of hydrogen-bond donors and acceptors, the presence of metal ions, the need for adaptive changes in conformation, and the lipophilicity of residues at the protein-ligand interface. Protein-protein interfaces of multiprotein assemblies share many of these undesirable features, although those that involve concerted binding and folding in their assembly have better defined pockets or grooves, and these can provide opportunities for identifying hits and for lead optimization. In some protein-protein interfaces conformational changes—often involving rearrangement of large side chains such as those of tyrosine, tryptophan, or arginine—are required to configure an appropriate binding site, and this may require tethering of the ligands until higher affinity is achieved. In many enzymes, larger conformational rearrangements are required to form the binding site, and these can make fragment-based approaches particularly difficult

Ulla: a program for calculating environment-specific amino acid substitution tables

Summary: Amino acid residues are under various kinds of local environmental restraints, which influence substitution patterns. Ulla,1 a program for calculating environment-specific substitution tables, reads protein sequence alignments and local environment annotations. The program produces a substitution table for every possible combination of environment features. Sparse data is handled using an entropy-based smoothing procedure to estimate robust substitution probabilities

Phosphopeptide interactions with BRCA1 BRCT domains: More than just a motif.

BRCA1 BRCT domains function as phosphoprotein-binding modules for recognition of the phosphorylated protein-sequence motif pSXXF. While the motif interaction interface provides strong anchor points for binding, protein regions outside the motif have recently been found to be important for binding affinity. In this review, we compare the available structural data for BRCA1 BRCT domains in complex with phosphopeptides in order to gain a more complete understanding of the interaction between phosphopeptides and BRCA1-BRCT domains.We thank Dr Takashi Ochi for helpful discussion and comments on the manuscript. QW and TLB are funded by the Wellcome Trust (Grant: 093167/Z/10/Z). HJ thanks UCB and the Biotechnology and Biological Sciences Research Council (BBSRC) for a CASE Studentship.This is the final published version. It first appeared at http://dx.doi.org/10.1016/j.pbiomolbio.2015.02.00

Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair.

Nonhomologous end joining (NHEJ) is central to the repair of double-stranded DNA breaks throughout the cell cycle and plays roles in the development of the immune system. Although three-dimensional structures of most components of NHEJ have been defined, those of the catalytic region of DNA ligase IV (LigIV), a specialized DNA ligase known to work in NHEJ, and of Artemis have remained unresolved. Here, we report the crystal structure at 2.4 Å resolution of the catalytic region of LigIV (residues 1-609) in complex with an Artemis peptide. We describe interactions of the DNA-binding domain of LigIV with the continuous epitope of Artemis, which, together, form a three-helix bundle. A kink in the first helix of LigIV introduced by a conserved VPF motif gives rise to a hydrophobic pocket, which accommodates a conserved tryptophan from Artemis. We provide structural insights into features of LigIV among human DNA ligases

SDM—a server for predicting effects of mutations on protein stability and malfunction

The sheer volume of non-synonymous single nucleotide polymorphisms that have been generated in recent years from projects such as the Human Genome Project, the HapMap Project and Genome-Wide Association Studies means that it is not possible to characterize all mutations experimentally on the gene products, i.e. elucidate the effects of mutations on protein structure and function. However, automatic methods that can predict the effects of mutations will allow a reduced set of mutations to be studied. Site Directed Mutator (SDM) is a statistical potential energy function that uses environment-specific amino-acid substitution frequencies within homologous protein families to calculate a stability score, which is analogous to the free energy difference between the wild-type and mutant protein. Here, we present a web server for SDM (http://www-cryst.bioc.cam.ac.uk/~sdm/sdm.php), which has obtained more than 10 000 submissions since being online in April 2008. To run SDM, users must upload a wild-type structure and the position and amino acid type of the mutation. The results returned include information about the local structural environment of the wild-type and mutant residues, a stability score prediction and prediction of disease association. Additionally, the wild-type and mutant structures are displayed in a Jmol applet with the relevant residues highlighted

CHOPIN: a web resource for the structural and functional proteome of Mycobacterium tuberculosis.

Tuberculosis kills more than a million people annually and presents increasingly high levels of resistance against current first line drugs. Structural information about Mycobacterium tuberculosis (Mtb) proteins is a valuable asset for the development of novel drugs and for understanding the biology of the bacterium; however, only about 10% of the ∼4000 proteins have had their structures determined experimentally. The CHOPIN database assigns structural domains and generates homology models for 2911 sequences, corresponding to ∼73% of the proteome. A sophisticated pipeline allows multiple models to be created using conformational states characteristic of different oligomeric states and ligand binding, such that the models reflect various functional states of the proteins. Additionally, CHOPIN includes structural analyses of mutations potentially associated with drug resistance. Results are made available at the web interface, which also serves as an automatically updated repository of all published Mtb experimental structures. Its RESTful interface allows direct and flexible access to structures and metadata via intuitive URLs, enabling easy programmatic use of the models.This work was supported by the Bill & Melinda Gates Foundation (RG60453). University of Cambridge for facilities and support [to TLB]. Funding for open access charge: Bill & Melinda Gates Foundation.This is the final published version. It first appeared at http://database.oxfordjournals.org/content/2015/bav026.long

Flexibility and small pockets at protein-protein interfaces: New insights into druggability.

The transient assembly of multiprotein complexes mediates many aspects of cell regulation and signalling in living organisms. Modulation of the formation of these complexes through targeting protein-protein interfaces can offer greater selectivity than the inhibition of protein kinases, proteases or other post-translational regulatory enzymes using substrate, co-factor or transition state mimetics. However, capitalising on protein-protein interaction interfaces as drug targets has been hindered by the nature of interfaces that tend to offer binding sites lacking the well-defined large cavities of classical drug targets. In this review we posit that interfaces formed by concerted folding and binding (disorder-to-order transitions on binding) of one partner and other examples of interfaces where a protein partner is bound through a continuous epitope from a surface-exposed helix, flexible loop or chain extension may be more tractable for the development of "orthosteric", competitive chemical modulators; these interfaces tend to offer small-volume but deep pockets and/or larger grooves that may be bound tightly by small chemical entities. We discuss examples of such protein-protein interaction interfaces for which successful chemical modulators are being developed.We thank our colleagues Alicia Higueruelo, Douglas Pires, Bernardo Ochoa and Chris Radoux for helpful comments and discussions. D.B.A is the recipient of a C. J. Martin Research Fellowship from the National Health and Medical Research Council of Australia (APP1072476). H.J. is supported by a CASE Studentship from the UCB and the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant: BB/J500574/1). T.L.B. receives funding from University of Cambridge and The Wellcome Trust for facilities and support.This is the accepted manuscript of a paper published in Progress in Biophysics and Molecular Biology (Jubb H, Blundell TL, Ascher DB, Progress in Biophysics and Molecular Biology 2015, doi:10.1016/j.pbiomolbio.2015.01.009). The final version is available at http://dx.doi.org/10.1016/j.pbiomolbio.2015.01.009

Model-building strategies for low-resolution X-ray crystallographic data

Interpretation of low-resolution X-ray crystallographic data can prove to be a difficult task. The challenges faced in electron-density interpretation, the strategies that have been employed to overcome them and developments to automate the process are reviewed