Hajredini, F. | bioRxiv Science

bioRxiv2024

Sequence-dependent scale for translocon-mediated insertion of interfacial helices in membranes

Biological membranes consist of a lipid bilayer studded with integral and peripheral membrane proteins. Most -helical membrane proteins require protein-conducting insertases known as translocons to assist in their membrane insertion and folding. While the sequence-dependent propensities for a helix to either translocate through the translocon or insert into the membrane have been codified into numerical hydrophobicity scales, the corresponding propensity to partition into the membrane interface remains unraveled. By engineering diagnostic glycosylation sites around test peptide sequences inserted into a host protein, we devised a system that can differentiate between water-soluble, surface-bound, and transmembrane (TM) states of the sequence based on its glycosylation pattern. Using this system, we determined the sequence-dependent propensities for transfer from the translocon to a TM, interfacial or extramembrane space. UMAP analysis of a large collection of TM and water-soluble helices provide useful embeddings for analysis of these propensities and aid in understanding the physical properties and functions of antimicrobial, lytic, and fusogenic peptides.

molecular biology↗

bioRxiv2022

Conserved Link between Catalytic Site Interactions and Global Conformation in P-loop Enzymes

P-loop enzymes, ubiquitous in all of lifes domains and viruses, comprise a monophyletic group with pre-LUCA origins that have differentiated into several three-layered /{beta}/- sandwich domain families utilizing a basic {beta}- loop--{beta} structural module housing conserved nucleotide-binding Walker-A and Walker-B sequences. We have analyzed a large dataset of P-loop enzyme structures representing both their KG and ASCE branches as proxies for their sampled conformational landscapes. We developed a novel framework to correlate global conformations and local catalytic site geometry, specifically involving the Walker motifs, to identify conserved signatures despite substantial structural and functional diversity. Our results suggest that P-loop enzymes populate global states broadly classifiable as open or closed. In the closed states, that share similar overall geometries irrespective of family, key catalytic site residues are aligned to optimally engage the critical Mg2+ ion suggesting compatibility with the chemical step. These catalytic site interactions are disrupted in the open states resulting in the loss of the Mg2+- coordinating ability yielding conformations incapable of chemistry. In contrast to the closed states, open states are highly diverse, and this variability is facilitated by differential coupling of specific residues that are part of, or spatially proximal to, the Walker motifs with the clade-specific tertiary fold. We suggest that an essential feature in the activation and nucleotide exchange processes for all P-loop enzymes is the universal coupling between global closure and local reorganization of the catalytic site for efficient coordination of Mg2+ that carries a tightly associated cargo, the substrate NTP.

biochemistry↗