Biology subjects

Grishaev, A.

Publications and source records attributed to Grishaev, A..

3 recordsLinked to original sources

bioRxiv2025

Conformational dynamics and multi-modal interaction of Paxillin with the Focal Adhesion Targeting Domain

Paxillin (PXN) and focal adhesion kinase (FAK) are two major components of the focal adhesion complex, a multiprotein structure linking the intracellular cytoskeleton to the cell exterior. The interaction between the disordered N-terminal domain of PXN and the C-terminal targeting domain of FAK (FAT) is necessary and sufficient for localizing FAK to focal adhesions. Furthermore, PXN serves as a platform for recruiting other proteins that together control the dynamic changes needed for cell migration and survival. Here, we show that the PXN N-domain undergoes significant compaction upon FAT binding, forming a 48-kDa multi-modal complex with four major interconverting states. Although the complex is flexible, each state has unique sets of contacts involving disordered regions that are both highly represented in ensembles and conserved. PXN being a hub protein, the results provide a structural basis for understanding how shifts in the multi-state equilibrium (e.g. through ligand binding and phosphorylation) may rewire cellular networks leading to phenotypic changes. TeaserThe fuzzy complex between Paxillin and Focal adhesion targeting domain is resolved, with functional and translational implications.

biophysics↗

bioRxiv2019

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Targeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 [A]) and an asymmetric form (AsymCDTb; 2.84 [A]). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI. SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

biophysics↗

bioRxiv2018

PAGE4 and Conformational Switching: Insights from Molecular Dynamics Simulations and Implications for Prostate Cancer

Prostate-Associated Gene 4 (PAGE4) is a disordered protein implicated in the progression of prostate cancer. PAGE4 can be phosphorylated at two residue sites by Homeodomain-Interacting Protein Kinase 1 (HIPK1) to facilitate its binding to the Activator Protein-1 (AP-1) transcription factor. In contrast, a further hyperphosphorylation of PAGE4 by CDC-Like Kinase 2 (CLK2) reduces its binding affinity to AP-1, thus affecting the androgen receptor (AR) activity. Both SAXS and smFRET experiments have shown a structural expansion of PAGE4 upon hyperphosphorylation and a significant increase in size at its N-terminal half than that at its C-terminus. To understand the molecular mechanism underlying this structural transition, we performed a series of constant temperature molecular dynamics simulations using Atomistic AWSEM -- a multi-scale molecular model combining detailed atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interaction drives a transient formation of an N-terminal loop, which causes the change in size for different phosphorylated forms of PAGE4. Phosphorylation also changes the preference of secondary structure formation of PAGE4, which signifies a transition between states that display different degree of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions of different forms of PAGE4 with AP-1 and AR, a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4 and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching among different forms of PAGE4 may potentially affect the efficiency of therapeutic targeting of AR.

biophysics↗