More than a decade of research has been published with the aim of assessing the bind-ing specificities of PDZ domains. As has been discussed in section 5.2, specificity can only be assessed when comparing binding affinities. Thus, systematic determination of binding affinities of several peptides to several PDZ domains combined with structural analysis is likely to significantly contribute to our understanding of PDZ interaction specificities. However, only very few of such studies (including our article presented in chapter 9) have been published so far [10,81,82]. Maybe it is because of this lack of data that we still do not clearly understand the rules that define which PBM will bind to which PDZ domain in particular and how specific or promiscuous PDZ–peptide interactions are in general.
5.6.1. Troubles in classification of PDZ domains
As introduced in section 3.2.1, C-terminal PBMs are commonly grouped into three subclasses, based on the residue at peptide position -2. Many studies concentrated on the analysis of which PDZ domain residues define to which of these subclasses of peptides a PDZ domain will bind. Some studies suggest that only very few domain residues, especially at the first and fifth position of the α2 helix (see Figure 3.4), de-termine the class membership of a PDZ domain. Mutations at these positions have been shown to be sufficient to convert the class specificity of one PDZ into another (see section 3.2.1) [73]. Motivated by these observations, Bezprozvanny and Maximov [146]
proposed a classification of PDZ domains into 25 groups based on the chemical prop-erties of the residues at the first and fifth position of the α2 helix. Contradictory opinions have been obtained by other studies, namely from Vaccaro et al. [147], who presented evidence that subclass affiliation of PDZ domains is much more complex.
In an interesting correspondence, they provide examples like the first PDZ domain of MPDZ that based on its residues at the two helix positions, should bind class I peptides but has been found to bind class II ligands [148].
Overall, researchers agreed on that the classification of PDZ domains and/or ligands into three subclasses is very unlikely to sufficiently explain the binding preferences ob-served between PDZ domains and their ligands. By now, it is widely accepted that the last four to five peptide residues each contribute to PDZ domain binding. It has been suggested that the number of accepted residues at peptide position -1 may define the level of promiscuity of a given PDZ domain [82]. Based on preferences for residues at the last six peptide positions, 16 distinct specificity classes of PDZ domains have been defined using a large-scale data set on phage display-derived PDZ–peptide inter-actions [9]. Interesting findings on multiple specificities of PDZ domains have been published by Gfelleret al. [92]. They grouped phage display peptides obtained for one PDZ domain into subgroups based on sequence similarities. This revealed that PDZ domains can recognize ligands of different subgroups (others than the three canonical classes described) that are bound by the PDZ domain in structurally very different ways [92]. Velthuiset al. [56] applied a published PDZ interaction predictor for screen-ing a whole proteome for potential binders to PDZ domains. Based on the screenscreen-ing results, they concluded that PDZ domains bind with extensive overlap to sets of pep-tides. However, as discussed in our article presented in chapter 9 the predictor they used suffered from a high false positive rate (FPR), overall questioning their findings.
These various results raise the question about which criteria can be used to classify PDZ domains. Is it sufficient to know one physiological or artificial ligand to be able to assign a PDZ domain to a certain class? How physiologically relevant are overall such class assignments? Quite a few PDZ domains have been shown to be able to bind ligands from at least two peptide subclasses (see section 3.2.1). In a seminal work, Wiedemannet al. [81] provided evidence that class assignment of PDZ domains highly depends on the affinity range considered (see Figure 5.3). PDZ domains can be of dual
specificity if low binding affinities are accepted but still might display a clear preference for ligands of one subclass. In line with these findings, their data also suggests that the level of overlap between PDZ domains for sets of bound ligands highly depends on the affinity cuf-off considered with high affinity cut-offs leading to just little overlap and lower affinity boundaries producing substantial overlap [81]. Thus, instead of tempting to group PDZ domains into distinct classes it might be more relevant to consider a continuous specificity space of PDZ domains as it has been suggested after analysis of the first large-scale data set on physiological PDZ–peptide interactions published from MacBeath and co-workers [10].
Figure 5.3. Influence of binding affinity cut-offs on the level of specificity of PDZ domains.
Wiedemann et al. [81] predicted binding affinities for all possible 4 residue-long peptides towards three human PDZ domains. For three different affinity cut-offs (indicated above the table), the number of predicted binding peptides of class I, class II, and non class I/II for each PDZ were counted and are shown in the table. With weaker binding affinity cut-offs, the PDZ domains become less specific accepting peptides from all three categories. This figure has been extracted from [81].
5.6.2. The role of phage display for studying PDZ binding specificities Phage display has been recurrently used to study the binding profiles and specificities of PDZ domains. The Sidhu lab developed and refined the protocol that allowed presenta-tion of C-terminal peptides by bacterial phages, and consequently applied it in several single case [60, 63, 82, 149–151] and large-scale studies on PDZ domains [9, 152, 153].
In phage display, billions of different peptides are expressed on the surface of bacte-riophages and are presented to an analyte (e.g. a PDZ domain) that is attached to a solid support. In several rounds of binding, washing, and amplification, peptides are selected that bind with high affinity to the analyte. The binding profile of the analyte can be obtained from consequent sequencing and alignment of the selected peptides.
Sidhu and co-workers often combined phage display with structural analysis and these studies have provided important contributions to our understanding of binding speci-ficities of PDZ domains.
Nevertheless, the standard phage display procedure has a major drawback that un-fortunately, is recurrently disregarded during data analysis. Peptide selection in phage
display is solely based on the binding affinities of the peptides to the presented analyte and their rates of dissociation. In contrast, cellular PBMs are selected by evolution for their functionality, e.g. mediating protein interactions in the signalling context.
Here, specific and transient interactions are observed and high binding affinity plays rather a secondary role. As a consequence, phage display peptides have sometimes been shown to bind with higher affinity to PDZ domains and to display different se-quences as compared to their natural counterparts [82,149,154] (this has been subject in our article presented in chapter 8). Thus, assessment of binding specificities of PDZ domains using binding profiles derived from phage display data can lead to results that are irrelevant in the biological context. Phage display data is currently the prevailing type of interaction data for PDZ domains that influenced much our understanding of PDZ–peptide interactions and the development of PDZ interaction predictors (see section 6.4.4). Care should be taken to properly interpret the data in its context and to prevent it from biasing our knowledge on PDZ-peptide interaction specificities.
5.6.3. Dynamic aspects of PDZ interaction specificities
Interesting insights in PDZ–peptide binding specificities have also been gained from molecular dynamics simulations. Nonpolar contacts have been observed to substan-tially contribute to the overall free energy of binding to PDZ domains [155]. It has been hypothesised that this might partially explain their observed promiscuous bind-ing behaviours [155]. The entropic contribution to PDZ bindbind-ing has often been un-favourable in molecular dynamics simulations [155]. Indeed, the peptide undergoes considerable structuring upon binding. PDZ domains have been observed to some-times become more rigid, somesome-times more flexible upon binding depending on the ligand studied. Thus, entropy might be an important player in binding specificities of PDZ domains [155]. The energetic contribution of water release upon peptide binding to PDZ domains has been studied more in detail by Beuming et al. [156]. Calcula-tions suggested that peptides displayed higher affinity to PDZ domains when they had residues, such as Trp, that displaced more water molecules [156]. However, it does not seem that such hydrophobicity-driven affinity changes necessarily lead to higher interaction specificities (see the discussion in our article presented in chapter 8).
5.6.4. Influence of sequence context on PDZ interaction specificities Most experimental studies on PDZ–peptide binding specificities used constructs com-prising the core PDZ domain and peptides of five residues in length. As discussed in chapters 9 and 10, binding affinities and specificities observed for such minimal interacting fragments can change when extending the constructs. Peptide residues upstream of the last five residues can modulate binding affinities and specificities to PDZ domains. Extensions of PDZ domains and neighbouring domains can alter the binding properties of the PDZ towards C-terminal ligands.
PDZ domains actually constitute a prototype for the study of the influence of such sequence context on the structure and function of a globular domain. For many PDZ domains it has been shown that they possess additional secondary structure elements that precede or follow the core PDZ fold and that impact the structure and function of the domain. Fewer examples exist where such extensions are disordered (see our NMR study presented in chapter 7). Neighbouring domains have also been demonstrated to influence significantly the fold and peptide binding properties of PDZ domains.
This is true for neighbouring domains that are non-PDZ domains, such as SH3 do-mains [15, 157] (see Figure 5.1), but has been particular striking for PDZ dodo-mains that are separated by just a short linker sequence (see Figure 5.4). Structural studies revealed that PDZ1 and PDZ2 of DLG proteins, PDZ4 and PDZ5 of PATJ, PDZ1 and PDZ2 as well as PDZ4 and PDZ5 of GRIP1, and PDZ1 and PDZ2 of amyloid beta A4 precursor protein-binding family A member 1 (APBA1) display many interdomain contacts and actually form one globular unit, thus the term supramodule has been introduced to describe them [158].
PDZ–peptide interactions found to be promiscuous when studiedin vitro with min-imal interacting fragments, might turn out to be more specific in a full length and in vivo context.
C-ter extension of PDZ5
Figure 5.4. The PDZ45 supramodule of PATJ.PDZ4 (green) and PDZ5 (orange) of PATJ form a supramodular structure. PDZ5 is in complex with a C-terminal PBM derived from NG2 (dark blue). Both PDZ domains interact with each other via multiple contacts including an unstructured C-terminal extension of PDZ5. The peptide binding pocket of PDZ4 (indicated by a black line) is inaccessible for PBMs due to the orientation of PDZ4 towards PDZ5 (PDB code: 3R0H [117]).
The reader will first be introduced to a widely used concept for assessing prediction performances before presenting different approaches for domain–motif interaction pre-dictions in general and PDZ domain-mediated interaction prepre-dictions in particular.
The terms defined in the following section are recurrently used in this chapter.