Reassessing a sparse energetic network within a single protein domain

Abstract

Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295-299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery.allostery ͉ coupling energy ͉ dynamics ͉ energetic network of residues ͉ PDZ domain P redicting allosteric mechanisms from protein sequence is an important goal, yet very difficult because the molecular basis for allostery in proteins is still not fully understood. Allosteric regulation is the alteration of protein function through the binding of an effector molecule elsewhere within the same protein. This definition can be expanded to include amino acid point mutations distal from the active site that can effectively alter protein function (1). Allosteric regulation is well established in multidomain proteins (2, 3), but allosteric behavior in single protein domains is less documented (for example, ref. 4), and its mechanisms unclear. Nevertheless, the idea of allostery without a (well defined) conformational change (5) has become popular, and it has even been proposed that all dynamic proteins are allosteric (6).Lockless and Ranganathan (7) set out to investigate whether allostery could be predicted from sequence conservation. They found a coevolved network of residues in the PDZ domain family of proteins, and these statistically coupled residues were confirmed by experiments on PSD95 PDZ3 to be energetically coupled (7). This study has served as a classic example of allostery in a single protein domain, with a ''sparse network'' of energetically linked positions that could affect function, in this case ligand binding. However, pathways of energetic connectivity inferred from statistical analysis have been questioned because the correlated mutation algorithm that was used finds pairs of residues that are close in physical space in the protein structure and it is therefore not surprising that the residues actually do couple (8). Furthermore, other issues regarding the validity of the statistical method used by Lockless and Ranganathan have been raised: the algorithm used is not symmetric (9), does not incorporate evolutionary noise (10), and performs less well than other algorithms (8,9,11). From an experimental point of view, previous work on PSD95 PDZ3 in our laboratory suggested that this PDZ ...

Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding

“…The numbering of residues refers to full length PSD-95α without exon b and is the same as used in Doyle et al 13 The F337W mutation serves as a fluorescence probe in ligand binding experiments and does not affect the affinity of the PDZ3-peptide interaction. [16][17][18] It also does not affect the energetic coupling pattern between side-chain residues in the PDZ domain and in the peptide ligand 9 , since the coupling energy for F337W is zero with regard to a second mutation in the peptide ligand, either at the C-terminal Val 0 or at Ser -2 (not shown). The sequence corresponding to the α3-helix (residues 396-401) was deleted from the cDNA of PDZ3 to generate the α3-helix-deleted mutant PDZ3∆α3.…”

Long-Range Conformational Transition of a Photoswitchable Allosteric Protein: Molecular Dynamics Simulation Study

“…16 The nature of the subsequent ligand-induced intramolecular signaling process, however, remains a matter of debate, although various proposals of signaling pathways exist. [30][31][32][33][34][35][36][37][38] To facilitate a dynamic perspective of the allosteric mechanism, recently Buchli et al 45 presented a time-resolved study of the transition from the free to the bound state of PDZ2 triggered by a molecular photoswitch. [46][47][48][49][50][51] By covalently linking an azobenzene photoswitch across the binding groove and using a femtosecond laser pulse that affects the cis → trans photoisomerization of azobenzene, they were able to initiate a conformational change similar to the free-bound transition.…”

Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

“…All mutants were made by PCR using Pfu Turbo polymerase (Stratagene) and the pseudo-wild-type PDZ3 F337W cDNA as template (21,36). This construct codes for residues 309 -401 of PSD-95 (compare Protein Data Bank file 1BFE; ref.…”