Biology subjects

Marlovits, T. C.

Publications and source records attributed to Marlovits, T. C..

5 recordsLinked to original sources

bioRxiv2021

Structural snapshots of human PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes

The uptake of peptides in mammals plays a crucial role in nutrition and inflammatory diseases. This process is mediated by promiscuous transporters of the Solute Carrier Family 15, which form part of the Major Facilitator superfamily. Besides the uptake of short peptides, Peptide transporter 1 (PepT1) is a highly abundant drug transporter in the intestine and represents a major route for oral drug delivery. Peptide transporter 2 (PepT2) allows in addition renal drug reabsorption from ultrafiltration and brain-to-blood efflux of neurotoxic compounds. Here we present cryo-EM structures of human PepT1 in an outward open state and of human PepT2 in an inward facing partially occluded state with a bound substrate. The structures reveal the architecture of human peptide transporters and provide mechanistic insights into substrate recognition and conformational transitions during transport. Importantly, this may support future drug design efforts to increase the bioavailability of different drugs in the human body.

biophysics↗

bioRxiv2020

Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation

Many bacterial pathogens strictly rely on the activity of type III secretion systems (T3SSs) to secrete and translocate effector proteins in order to establish infection. The central component of T3SSs is the needle complex, a supramolecular machine which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to allow bacterial effectors to gain entry into the host cell cytoplasm to modulate signal transduction processes. Disruption of this process impairs pathogenicity, providing an avenue for antimicrobial design. However, the molecular principles underlying T3 secretion remain elusive. Here, we report the first structure of an active Salmonella enterica sv. Typhimurium needle complex engaged with the late effector protein SptP in two functional states, revealing the complete 800[A]-long secretion conduit and unravelling the critical role of the export apparatus (EA) subcomplex in T3 secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent transport, explaining heterogeneity and structural disorder of signal sequences in T3SS effector proteins. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates but prevents leaky pore formation to maintain the physical boundaries of compartments separated by a biological membrane. Following gate penetration, a moveable SpaR loop first folds up to then act akin to a linear ratchet to steer substrates through the needle complex. Together, these findings establish the molecular basis for substrate translocation through T3SSs, improving our understanding of bacterial pathogenicity and motility of flagellated bacteria, and paves the way for the development of novel concepts combating bacterial infections.

molecular biology↗

bioRxiv2020

Structure and dynamics of the ESX-5 type VII secretion system of Mycobacterium tuberculosis

Mycobacterium tuberculosis causes one of the most important infectious diseases in humans, leading to 1.5 million deaths every year. Specialized protein transport systems, called type VII secretion systems (T7SSs), are central for its virulence, but also crucial for nutrient and metabolite transport across the mycobacterial cell envelope. Here we present the first structure of an intact T7SS inner membrane complex of M. tuberculosis. We show how the 2.32 MDa, 165 transmembrane helices-containing ESX-5 assembly is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber formed by three EccB5 dimers, with the proteolytic sites facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, highlighting the importance of this component for complex integrity. Beneath the EccB5-MycP5 chamber, dimers of the EccC5 ATPase assemble into three four-transmembrane helix bundles, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations, likely reflecting different secretion states. Our work suggests a novel mechanism of protein transport and provides a structural scaffold to aid drug development against the major human pathogen.

microbiology↗

bioRxiv2020

Functional self-nonamerization of the Type III translocase chaperone/exported protein receptor

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante- chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E.coli (EPEC). In both EPEC and E.coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of [~]22 nm with a N- terminal domain connected by a short linker to a C-domain ring structure with a [~]5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 [A] and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with pore surface and ring entry mouth local conformational changes that are also modulated by the ATPase inner stalk. We propose a model that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.

molecular biology↗

bioRxiv2019

Structural control for the coordinated assembly into functional pathogenic type-3 secretion systems

Functional injectisomes of the type-3 secretion system assemble into highly defined and stoichiometric bacterial molecular machines essential for infecting human and other eukaryotic cells. However, the mechanism that governs the regulated step-wise assembly process from the nucleation-phase, to ring-assembly, and the filamentous phase into a membrane embedded needle complex is unclear. We here report that the formation of a megadalton-sized needle complexes from Salmonella enterica serovar Typhimurium (SPI-1, Salmonella pathogenicity island-1) with proper stoichiometries is highly structurally controlled competing against the self-assembly propensity of injectisome components, leading to a highly unusual structurally-pleiotropic phenotype. The structure of the entire needle complex from pathogenic injectisomes was solved by cryo electron microscopy, focused refinements (2.5-4 [A]) and co-variation analysis revealing an overall asymmetric arrangement containing cyclic, helical, and asymmetric sub-structures. The centrally located export apparatus assembles into a conical, pseudo-helical structure and provides a structural template that guides the formation of a 24-mer cyclic, surrounding ring, which then serves as a docking interface comprising three different conformations for sixteen N-terminal InvG subunits of the outer secretin ring. Unexpectedly, the secretin ring excludes the 16th protein chain at the C-terminal outer ring, resulting in a pleiotropic 16/15-mer ring and consequently to an overall 24:16/15 basal body structure. Finally, we report how the transition from the pseudo-helical export apparatus into the helical filament is structurally resolved to generate the protein secretion channel, which provides the structural basis to restrict access of unfolded effector substrates. These results highlight the diverse molecular signatures required for a highly coordinated assembly process and provide the molecular basis for understanding triggering and transport of unfolded proteins through injectisomes.

molecular biology↗