We have introduced comparative patch analysis, an approach to the modeling of a complex between two subunit structures, and applied it to the protein PSD-95, a key neural-signaling scaffold. The approach relies on structurally defined interactions of each of the complex components, or their homologs, with any other subunit, irrespective of its fold (). We assessed comparative patch analysis for its increased applicability relative to comparative modeling as well as increased accuracy relative to conventional protein docking (, ). Next, comparative patch analysis was applied to model the structure of a core fragment of rat PSD-95, containing the PDZ3, SH3, and GK domains, resulting in two predicted configurations ( and ). The model was experimentally supported by limited proteolysis (). In addition, the prediction is in concordance with and rationalizes available biochemical, structural, and evolutionary data ( and , ).
Comparative Patch Analysis
By limiting the configurational search to the known binding modes of the homologous subunits and applying a physical assessment of candidate complex structures, comparative patch analysis benefits from the advantages of both homology-driven and physics-driven docking. Its coverage is larger than that of comparative modeling and its accuracy is higher than that of protein docking (), although the coverage and accuracy are lower than those of protein docking and comparative modeling, respectively.
At least one binding site is available for 1,989 of the 3,114 total Structural Classification of Proteins (SCOP) domain families (release 1.69, July 2005). Eight hundred fifty of these families contain between ten and 100 binding sites, allowing the exhaustive pairwise docking that is currently required. Thus, the applicability of comparative patch analysis extends to approximately 41%, and in the current implementation is computationally feasible for 8%, of the ~4,850,000 theoretically possible binary domain–domain interactions. The coverage of conventional protein docking is 100%, while the comparative modeling approach is applicable to only 2,126 pairs of families, which constitutes 0.06% of the theoretically possible interactions.
When compared with protein docking, comparative patch analysis was able to correctly identify the binding mode in 40% more benchmark complexes, predicting the overall structure of the complexes with an average improvement in all-atom RMS error of 13.4 Å. The method also exhibits robustness to small errors in the locations of the specified binding sites, due to the configurational search performed by the docking procedure. In the benchmark set of complexes with known structures, a minimal threshold of 75% overlap between the initially specified and resulting refined binding sites captured all but one of the good models (LRMS error less than 3 Å), while allowing no false positives.
PSD-95 Protein: Predicting the Structure of the Core Fragment by Analogy
Evolutionary and experimental evidence for intermolecular interaction between PDZ3 and SH3–GK domains.
When modeling the structure of the PDZ3–SH3–GK fragment, we assumed an interaction between the PDZ3 and SH–GK domains. PDZ3 is a good candidate for interaction with the SH3–GK domains because it is immediately upstream of SH3, separated by a relatively short 14-residue linker. To investigate whether or not PDZ3 interacts with SH3–GK, the analysis of domain co-occurrence, as well as limited proteolysis, were applied.
A survey of the domain architectures of proteins that contain both SH3 and GK domains revealed that the proteins either do not have other domains or also contain at least one PDZ domain always preceding the SH3–GK tandem domain. The minimal architecture that contains at least one PDZ, SH3, and GK domain consists of only these three domains. This pattern strongly suggests a physical interaction between the SH3–GK tandem and the preceding PDZ domain [51
The stable fragments resulting from limited proteolysis of PSD-95 by nonspecific proteases reflect the cleavage of accessible loops, rather than cleavage at a particular substrate sequence. We identified stable PDZ3–SH3–GK and SH3–GK fragments by mass spectrometry, demonstrating susceptibility of PSD-95 to protease cleavage at sites between the PDZ2 and PDZ3 domains and between the PDZ3 and SH3–GK domains. Limited proteolysis with trypsin (unpublished data) also supports the conclusion that the PDZ3 and SH3–GK domains are stable protein structures. These data are consistent with intramolecular interactions between the PDZ3 and the SH3–GK domains of PSD-95.
Application of comparative patch analysis.
Modeling the structure of the core PSD-95 fragment is challenging for a number of reasons. First, the structures of neither PDZ–SH3 nor PDZ–GK complexes are available, rendering comparative modeling inapplicable in this case. Moreover, conventional protein docking results were ambiguous, generating a varied ensemble of PDZ3
and SH3–GK complexes without a predominant binding mode (C). On the other hand, each of the domain families is known to repeatedly utilize a small number of binding sites for different protein interactions. For instance, PDZ domains bind the C-termini of several different proteins through its hydrophobic cleft [42
]. Similarly, the PRBS of SH3 domains recognizes PXXP-sequence motifs in a variety of proteins [45
]. These observations suggest that comparative patch analysis is suited for modeling the PSD-95 core fragment.
Functional roles of the predicted configurations.
Comparative patch analysis of the PDZ3
–SH3–GK fragment found two possible configurations that satisfied all imposed spatial restraints, including previously observed binding sites, consistency with the given linker length, and physicochemical complementarity of the interacting surfaces. In addition, the ensemble of models produced by comparative patch analysis for each interaction type (PDZ3
–GK) exhibited a single predominant binding mode. The binding sites forming the interaction interfaces of these models are located at the same or similar regions of the protein surface (). Therefore, the binding modes are predicted with relative confidence. Multiple stable configurations of PSD-95 and its close homologs have recently been suggested independently based on biochemical studies [40
] and single-particle electron microscopy experiments [41
]. As we describe below, we suggest the two binding modes have clear functional implications.
The two predicted configurations exhibit structural properties that suggest unique functional roles. In the first configuration, the hydrophobic cleft of the PDZ domain and the GBS of the GK domain are both accessible, suggesting that this configuration corresponds to an active state in which binding of other proteins at these two sites can occur (B). These binding sites are thought to mediate intermolecular interactions essential for the scaffolding role of PSD-95 [42
]. In contrast, both binding sites are buried in the second configuration, by the interface between the PDZ3
and GK domains (C), which is suggestive of an alternative functional state. This second configuration points to an efficient intramolecular regulatory mechanism for switching the functional state with a single interaction. Similar regulatory mechanisms have been observed in other signaling networks, such as the TCR and MAPK systems [58
], indicating this regulation may be a general feature of signaling pathways.
This two-state model also provides a structural explanation for the change in binding affinity between the GK domain and MAP1A protein in the presence of the PDZ3
]. It has been shown that the GK domain alone is able to bind MAP1A. In the presence of PDZ3
, this binding affinity is dramatically reduced. The affinity is recovered upon titration of a C-terminal peptide of CRIPT known to specifically interact with the hydrophobic cleft of PDZ3
. This competitive binding suggests that binding to MAP1A and binding to PDZ3
are mediated by the same GK binding site. Our model is in complete agreement with this hypothesis and provides a structural explanation for these observations.
It is known that SH3 domains bind proteins with PXXP sequence motifs through their proline-rich binding regions. The proximity of the PDZ3
PXXP motif to the SH3 PRBS in the first configuration proposed by comparative patch analysis is consistent with the classical SH3–PXXP motif recognition. A similar PXXP-mediated intermolecular PDZ–SH3 interaction has been previously suggested to occur in syntenin [61
]. Sequence analysis of PSD-95 from different species indicates that PXXP motifs are not found in its other two PDZ domains, although such motifs are found in the PDZ2
linkers and the flexible N-terminus (). Recent studies have demonstrated the importance of disordered regions in binding events [62
], suggesting that future investigation of interactions of these PXXP motifs using recently developed flexible docking algorithms [63
] should prove fruitful.
The limited proteolysis experiment () is a first step to verifying the intramolecular interactions suggested by comparative patch analysis. The two functional states hypothesis, outlined in the Discussion
, points to a number of experiments that could shed light on the structure and function of PSD-95. First, the proposed regulation of the PSD-95 activity by PDZ3
–specific C-terminal peptides can be further tested using immunoprecipitation and yeast two-hybrid experiments similar to those performed for other GK-mediated interactions [60
] (e.g., with the GKAP protein [57
]). If the proposed regulation mechanism is verified, experimental control of the PSD-95 activity may become possible, enabling detailed study of the functional differences between the two states. Next, the intramolecular interactions proposed here can be tested by a variety of experimental techniques [64
], including NMR spectroscopy [65
], site-directed mutagenesis [66
], hydrogen/deuterium exchange combined with mass spectrometry [67
], and small angle X-ray scattering (SAXS) [68
]. In particular, site-directed mutagenesis [66
] of the interface residues in the first proposed state (see Datasets S1
) could be used with pull-down assays to validate the predicted interaction interface [69
]. In addition, the lack of accessibility of the GBS in the second state could be tested using nucleotide-binding assays [70
]. Finally, the shapes of the calculated SAXS spectra for the best-scoring models in both conformations are substantially different (). Thus, we expect the experimentally obtained SAXS spectra to be helpful in distinguishing between the two PSD-95 states.
Comparative patch analysis for characterizing the quaternary structure of protein assemblies provides a framework for combining data from known protein structures with a physical assessment of protein interactions. This framework will benefit from future developments in protein–protein docking, such as the explicit treatment of flexibility and more accurate scoring functions. We are currently developing an automated comparative patch analysis pipeline for large-scale modeling of protein complexes via a Web server. In closing, we expect that comparative patch analysis will provide useful spatial restraints for the structural characterization of an increasing number of binary and higher order protein complexes, as it did for PSD-95.