Biology subjects

Laurino, P.

Publications and source records attributed to Laurino, P..

22 records · Page 2Linked to original sources

bioRxiv2021

Sustained Enzymatic Activity and Flow in Crowded Protein Droplets

Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

biophysics↗

bioRxiv2021

Protein and substrate flexibility contribute to enzymatic specificity in human and bacterial methionine adenosyltransferases

Protein conformational change can facilitate the binding of non-cognate substrates and underlie promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here we analyse human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, non-cognate substrates bind in a stable conformation to allow catalysis. In contrast, non-cognate substrates rarely sample stable productive binding modes in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrate likely drove the evolutionary divergence of substrate specificity in these orthologs. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated level in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.

biochemistry↗

bioRxiv2020

A highly diverged ancient interface plays a key role in the origin and evolution of Methionine AdenosylTransferase superfamily

Methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine from L-methionine and ATP, is an ancient, highly conserved enzyme present in all three domains of life. Although the MAT enzymes of each domain are believed to share a common ancestor, the sequences of archaeal MATs show a high degree of divergence from the sequences of bacterial and eukaryotic MATs. However, the structural and functional consequences of this sequence divergence are not well understood. Here, we use structural bioinformatics analysis and ancestral sequence reconstruction to highlight the consequences of archaeal MAT divergence. We show that the dimer interface containing the active site, which would be expected to be well conserved across all three domains, diverged considerably between the bacterial/eukaryotic MATs and archaeal MATs. Furthermore, the characterization of reconstructed ancestral archaeal MATs showed that they probably had low substrate specificity which expanded during their evolutionary trajectory hinting towards the observation that all the modern day MAT enzymes from the three-kingdom probably originated from a common specific ancestor and then archaea MATs diverged in sequence, structure and substrate specificity. Altogether, our results show that the archaea MAT is an ideal system for studying an enzyme family which evolved to display high degrees of divergence at the sequence/structural levels and yet are capable of performing the same catalytic reactions as their orthologous counterparts.

evolutionary biology↗

bioRxiv2019

Epidermal Growth Factor (EGF) single mutants highlighted by a homologs cross-conservation approach differentially affect the EGF Receptor (EGFR) downstream pathway

The Epidermal Growth Factor Receptor (EGFR) is a membrane-anchored tyrosine kinase that is able to selectively respond to multiple extra-cellular stimuli. Previous studies have indicated that the modularity of this system is affected by ligand-induced differences in the stability of the dimerized receptor in a process known as "Biased signaling". However, this hypothesis has not been explored using single-mutant ligands thus far. Herein, we developed a new approach to identify residues responsible for functional divergence combining the conservation and co-evolution information of ortholog and paralog genes encoding the epidermal growth factor (EGF) ligand. Then, we mutated these residues and assessed the mutants effects on the receptor by employing a combination of molecular dynamics (MD) and biochemical techniques. Although the EGF mutants had comparable binding affinities to the wild type ligand for EGFR, the EGF mutants induced a different phosphorylation and cell growth pattern in multiple cell lines. The MD simulations of the EGF mutants show a long-range effect on the receptor dimer interface. For the first time in this study, a single mutation in EGF is shown to be enough to alter the activation of the pathway at the cellular level. These results also support the theory of biased signaling in the tyrosine kinase receptor system and demonstrate a promising new way to study ligand-receptor interactions.

bioinformatics↗