Biology subjects

Murphy, M. E. P.

Publications and source records attributed to Murphy, M. E. P..

3 recordsLinked to original sources

bioRxiv2021

Mechanism of the CBM35 domain in assisting catalysis by Ape1, a Campylobacter jejuni O-acetyl esterase

Peptidoglycan (PG) is O-acetylated by bacteria to resist killing by host lysozyme. During PG turnover, however, deacetylation is a prerequisite for glycan strand hydrolysis by lytic transglycosylases. Ape1, a de-O-acetylase from Campylobacter jejuni, is a bi-modular protein composed of an SGNH hydrolase domain and a CBM35 domain. The conserved Asp-His-Ser catalytic triad in the SGNH hydrolase domain confers enzymatic activity. The PG binding mode and function of the CBM35 domain in de-O-acetylation remained unclear. In this paper, we present a 1.8 [A] resolution crystal structure of a complex between acetate and Ape1. An active site cleft is formed at the interface of the two domains and two large loops from the CBM35 domain form part of the active site. Site-directed mutagenesis of residues in these loops coupled with activity assays using p-nitrophenol acetate indicate the CBM35 loops are required for full catalytic efficiency. Molecular docking of a model O-acetylated hexasaccharide PG substrate to Ape1 using HADDOCK suggests the interaction is formed by the active cleft and the saccharide motif of PG. Together, we propose that the active cleft of Ape1 diverges from other SGNH hydrolase members by using the CBM35 loops to assist catalysis. The concave Ape1 active cleft may accommodate the long glycan strands for selecting PG substrates to regulate subsequent biological events.

biochemistry↗

bioRxiv2020

Biophysical characterization of the ETV6 PNT domain polymerization interfaces

ETV6 is an ETS family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, we demonstrate that ETV6 PNT domain variants with monomerizing mutations adopt very stable helical bundle folds that do not change in conformation upon self-association. Amide hydrogen exchange and surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

biochemistry↗

bioRxiv2019

A Bacterial Surface Layer Protein Exploits Multi-step Crystallization for Rapid Self-assembly

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the C. crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multi-step assembly pathway. This pathway involves two domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2D crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using time-resolved electron cryo-microscopy (Cryo-EM) reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the two domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.\n\nSignificance StatementMany microbes assemble a crystalline protein layer on their outer surface as an additional barrier and communication platform between the cell and its environment. Surface layer proteins efficiently crystallize to continuously coat the cell and this trait has been utilized to design functional macromolecular nanomaterials. Here, we report that rapid crystallization of a bacterial surface layer protein occurs through a multi-step pathway involving a crystalline intermediate. Upon calcium-binding, sequential changes occur in the structure and arrangement of the protein, which are captured by time-resolved small angle x-ray scattering and transmission electron cryo-microscopy. We demonstrate that a specific domain is responsible for enhancing the rate of self-assembly, unveiling possible evolutionary mechanisms to enhance the kinetics of 2D protein crystallization in vivo.

biophysics↗