Uncovering protein structure

Structural biology is the study of the molecular arrangement and dynamics of biological macromolecules, particularly proteins. The resulting structures are then used to help explain how proteins function. This article gives the reader an insight into protein structure and the underlying chemistry and physics that is used to uncover protein structure. We start with the chemistry of amino acids and how they interact within, and between proteins, we also explore the four levels of protein structure and how proteins fold into discrete domains. We consider the thermodynamics of protein folding and why proteins misfold. We look at protein dynamics and how proteins can take on a range of conformations and states. In the second part of this review, we describe the variety of methods biochemists use to uncover the structure and properties of proteins that were described in the first part. Protein structural biology is a relatively new and exciting field that promises to provide atomic-level detail to more and more of the molecules that are fundamental to life processes

Uncovering protein function: from classification to complexes

Almost all interactions and reactions that occur in living organisms involve proteins. The various biological roles of proteins include, but are not limited to, signal transduction, gene transcription, cell death, immune function, structural support, and catalysis of all the chemical reactions that enable organisms to survive. The varied roles of proteins have led to them being dubbed 'the workhorses of all living organisms'. This article discusses the functions of proteins and how protein function is studied in a laboratory setting. In this article, we begin by examining the functions of protein domains, followed by a discussion of some of the major classes of proteins based on their function. We consider protein binding in detail, which is central to protein function. We then examine how protein function can be altered through various mechanisms including post-translational modification, and changes to environment, oligomerisation and mutations. Finally, we consider a handful of the techniques employed in the laboratory to understand and measure the function of proteins

Most yeast SH3 domains bind peptide targets with high intrinsic specificity

<div><p>A need exists to develop bioinformatics for predicting differences in protein function, especially for members of a domain family who share a common fold, yet are found in a diverse array of proteins. Many domain families have been conserved over large evolutionary spans and representative genomic data during these periods are now available. This allows a simple method for grouping domain sequences to reveal common and unique/specific binding residues. As such, we hypothesize that sequence alignment analysis of the yeast SH3 domain family across ancestral species in the fungal kingdom can determine whether each member encodes specific information to bind unique peptide targets. With this approach, we identify important specific residues for a given domain as those that show little conservation within an alignment of yeast domain family members (paralogs) but are conserved in an alignment of its direct relatives (orthologs). We find most of the yeast SH3 domain family members have maintained unique amino acid conservation patterns that suggest they bind peptide targets with high intrinsic specificity through varying degrees of non-canonical recognition. For a minority of domains, we predict a less diverse binding surface, likely requiring additional factors to bind targets specifically. We observe that our predictions are consistent with high throughput binding data, which suggests our approach can probe intrinsic binding specificity in any other interaction domain family that is maintained during evolution.</p></div

Specific conservation values for the yeast SH3 domain family.

<p>A. Alignment of the core 60 positions colored by ortholog SC values as a heat map (red high and yellow low SC values, with domains sorted alphabetically). The average SC value across the family is indicated for each position at the bottom of the table, along with the paralog positional entropy, surface labels and secondary structure. Dark Boxes indicate the 2 principal loop regions where high SC values are found. B. Specific conservation across the domain. The line is set at an SC value of 1.7, which is considered a potential threshold for significant specific conservation (where ortholog conservation is almost twice that of paralog conservation).</p

General mechanisms to obtain binding specificity in domain families.

<p>A. Domains may use the interaction with an extended region that goes beyond the canonical binding site to obtain intrinsic specificity (1). For example, the Abp1p SH3 domain binds extended target peptides (17 residues) and was shown to possess high intrinsic binding specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref009" target="_blank">9</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref010" target="_blank">10</a>]. Domains may also achieve intrinsic specificity through non-canonical recognition via an alternative binding surface far from the canonical one. For example, Pex13 is a peroxisomal membrane protein that contains an SH3 domain that binds Pex14p via the canonical binding surface, however, it also binds Pex5p through an alternative non-canonical surface [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref011" target="_blank">11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref012" target="_blank">12</a>]. Furthermore, intrinsic specificity may be achieved through replacing the canonical binding site with a non-canonical one (2) that would lead to negative selection (3) with respect to proline-rich peptides that bind SH3 domains. For example, Fus1 peptide targets do not contain a canonical PxxP motif thus minimizing cross reactivity to proline containing peptides [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref013" target="_blank">13</a>]. Some domains may have potential for contextual specificity using adjacent domains (4). For example, at least 2 of the 3 adjacent SH3 domains of Nck are required to bind their targets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref014" target="_blank">14</a>]. Spatial and temporal separation mechanisms may be another contextual specificity mechanism (6). For example, <i>in vitro</i>, Fyn SH3 domain and CD2BP2 both bind and compete with each other for the proline region in the target protein CD2. However CD2BP2 localizes to the cytosolic compartment where it interacts with CD2 in T-cells, while Fyn is present permanently in the lipid raft fraction unable to compete [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref015" target="_blank">15</a>]. In some cases, both intrinsic and contextual specificity mechanisms may be used by a domain, such as the Pex13p example above (5). We note here that contextual specificity has been used elsewhere to mean the extended regions of SH3 domain binding peptides, outside their core binding motif [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref016" target="_blank">16</a>]. This definition does not pertain to contextual specificity as discussed within this study. Figure adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref017" target="_blank">17</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref007" target="_blank">7</a>]. B. An example of an extended peptide-domain interaction. The Ark1 peptide is represented in stick and the SH3 domain from Abp1 uses space-filling. The red region is surface I and the blue region is surface II. W36 is represented as green and is on the boundary of the two surfaces. Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref018" target="_blank">18</a>] (pdb code 2rpn).</p

Example sequence conservation analysis for orthologs of Abp1 SH3 domain.

<p>The residues are colored according to the residue equivalence groups defined for entropy and PSSM calculations. The species names end with a number that refers to their taxonomic group (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.s003" target="_blank">S1 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.s001" target="_blank">S1 Table</a>). The SC value is calculated as (paralog entropy)/(ortholog entropy). A standard numbering system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.ref046" target="_blank">46</a>] for the core 60 SH3 domain residues is indicated on the top row as well as the residue number in the full length <i>S.cerevisiae</i> protein (fifth row). The paralog entropy is calculated from an alignment of the 28 SH3 domains in <i>S.cerevisiae</i>.</p

SI and SII PSSM for yeast paralog alignment (28 domains) and example ortholog alignments for Fus1 (16 species) and Bud14 (29 species).

<p>Total occurrence for each amino acid group for each position is indicated and colored as yellow (low) to red (high). Residues are grouped into SI (left) and SII (right). Dark outlined regions indicate most common preference for the family (≥ 20 occurrences). Overall, for SI there is a family preference for aromatic residues except the less conserved positions 9, 52 and 53. Notable exceptions include Fus1 that has cysteines at positions 37 and 54 (which are usually in the FWYH group). For SII, there is a loose family preference for polar/acidic residues except at position 49 where hydrophobic residues are found. The extent of conservation in the orthlog alignments in SI and SII vary, with a much greater variation seen in SII PSSMs. PSSMs for all domains (showing both complete domain sequence and only surface I/II) can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193128#pone.0193128.s008" target="_blank">S4 File</a>.</p

SI and SII family dendograms.

<p>Clustering was based on SI (left) and SII (right) PSSMs. For SI dendogram, there is more significant clustering, which appears to concentrate domains that bind class I peptides into the red group and domains that bind class II peptides into the magenta group.</p

A disordered encounter complex is central to the yeast Abp1p SH3 domain binding pathway

Protein-protein interactions are involved in a wide range of cellular processes. These interactions often involve intrinsically disordered proteins (IDPs) and protein binding domains. However, the details of IDP binding pathways are hard to characterize using experimental approaches, which can rarely capture intermediate states present at low populations. SH3 domains are common protein interaction domains that typically bind proline-rich disordered segments and are involved in cell signaling, regulation, and assembly. We hypothesized, given the flexibility of SH3 binding peptides, that their binding pathways include multiple steps important for function. Molecular dynamics simulations were used to characterize the steps of binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA. Before binding, the N-terminal segment 1 of ArkA is pre-structured and adopts a polyproline II helix, while segment 2 of ArkA (C-terminal) adopts a 310 helix, but is far less structured than segment 1. As segment 2 interacts with AbpSH3, it becomes more structured, but retains flexibility even in the fully engaged state. Binding simulations reveal that ArkA enters a flexible encounter complex before forming the fully engaged bound complex. In the encounter complex, transient nonspecific hydrophobic and long-range electrostatic contacts form between ArkA and the binding surface of SH3. The encounter complex ensemble includes conformations with segment 1 in both the forward and reverse orientation, suggesting that segment 2 may play a role in stabilizing the correct binding orientation. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to the fully engaged state. In this transition, ArkA makes specific contacts with AbpSH3 and buries more hydrophobic surface. Simulating the binding between ApbSH3 and ArkA provides insight into the role of encounter complex intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs in general