Khare, S. | Search bioRxiv

bioRxiv2018

Dissection of protonation sites for antibacterial recognition and transport in QacA, a multi-drug efflux transporter.

QacA is a drug:H+ antiporter (DHA2) with 14 transmembrane helices, that renders antibacterial resistance to methicillin-resistant Staphylococcus aureus (MRSA) strains, with homologues in other pathogenic organisms. It is a highly promiscuous antiporter, capable of H+- driven efflux of a wide array of cationic antibacterial compounds and dyes. Our study, using a homology model of QacA, reveals a group of six protonatable residues in its vestibule. Systematic mutagenesis resulted in identification of D34 (TM1), and a cluster of acidic residues in TM13 including E407 and D411 and D323 in TM10, as being crucial for substrate recognition and transport of monovalent and divalent cationic antibacterial compounds. The transport and binding properties of QacA and its mutants were explored using whole cells, inside-out vesicles, substrate-induced H+ release and microscale thermophoresis. We identify two sites, D34 and D411 as vital players in substrate recognition while E407 facilitates substrate efflux as a protonation site. We also observe that E407 plays a moonlighting role as a substrate recognition site for dequalinium transport. These observations rationalize the promiscuity of QacA for diverse substrates. The study unravels the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.\n\nHighlightsO_LIA homology model of QacA with 14 TM helices was used to test the importance of acidic residues within the vestibule.\nC_LIO_LIMono and Divalent cationic substrate recognition requires two sites D34 (TM1) and D411 (TM13).\nC_LIO_LIE407 (TM13) is important for protonation driven efflux.\nC_LIO_LISubstrate recognition of a divalent substrate, dequalinium occurs at E407 instead of D411 providing glimpses into the promiscuity of substrate recognition in QacA.\nC_LI

biochemistry↗

bioRxiv2018

Stimulus-responsive self-assembly of enzymatic fractal structures by computational design

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns at increasing length scales in fractal-shaped branched objects, e.g., trees, lungs, and sponges, results in high effective surface areas, and provides key functional advantages, e.g., for molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies will provide access to novel classes of functional biomaterials for wide ranging applications. Here, we describe a modular, multi-scale computational design method for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Computationally-guided atomic-resolution modeling of fusions of symmetric, oligomeric proteins with Src homology 2 (SH2) binding domain and its phosphorylatable ligand peptide was used to design iterative branching leading to fractal-like assembly formation by enzymes of the atrazine degradation pathway. Structural characterization using various microscopy techniques and Cryo-electron tomography revealed a variety of dendritic, hyperbranched, and sponge-like topologies which are self-similar over three decades ([~]10nm-10m) of length scale, in agreement with models from multi-scale computational simulations. We demonstrate control over mesoscale topology (by linker design), formation dynamics, and functional enhancements due to dynamic multi-component assemblies constructed with three atrazine degradation pathway enzymes. The described design method should enable the construction of a variety of novel, spatiotemporally responsive catalytic biomaterials featuring fractal topologies.

biophysics↗