Biology subjects

Smit, J. H.

Publications and source records attributed to Smit, J. H..

6 recordsLinked to original sources

bioRxiv2022

Evolutionary adaptation of the folding pathway for secretability

Secretory preproteins of the Sec pathway bear signal peptides and are targeted post-translationally to cross the plasma membrane or ER through translocases. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or after further trafficking. During cytoplasmic transit, mature domains must remain non-folded for translocase recognition and translocation. Here, we sought the structural basis for the delayed folding mechanism of mature domains and how this is regulated by signal peptides. To address this, we compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural twin cytoplasmic PpiB. Using global and local hydrogen deuterium exchange mass spectrometry we showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, that displayed different order and rates. Folding is delayed in PpiA by less hydrophobic/bulky native contacts, frustrated residues and a critical {beta} -turn in the early folding region and by signal peptide-driven disorder, which disrupts foldon hierarchy. When selected PpiA residues and its signal peptide were grafted onto PpiB they converted it into a slow folder with enhanced in vivo secretion. These data reveal the structural basis of non-folding in a secretory protein, that allows its trafficking.

biochemistry↗

bioRxiv2021

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the {gamma}-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptide promotes clamp closing; their mature domain overcomes the rate limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly catch and release trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics and become secreted.

biochemistry↗

bioRxiv2021

A nexus of intrinsic dynamics underlies translocase priming

The cytoplasmic ATPase SecA and the membrane-embedded SecYEG channel assemble to form the functional Sec translocase. How this interaction primes and catalytically activates the translocase remains unclear. We now show that priming exploits a sophisticated nexus of intrinsic dynamics in SecA. Using atomistic simulations, single molecule FRET and hydrogen/deuterium exchange mass spectrometry we reveal multiple distributed dynamic islands that cross-talk with domain and quaternary motions. These dynamic elements are highly conserved and essential for function. Central to the nexus is a slender Stem through which, motions in the helicase ATPase domain of SecA biases how the preprotein binding domain rotates between open-closed clamping states. Multi-tier dynamics are enabled by an H-bonded framework covering most of the SecA structure and allowing conformational alterations with minimal energy inputs. As a result, dimerization, the channel and nucleotides select pre-existing conformations, and alter local dynamics to restrict or promote catalytic activity and clamp motions. These events prime the translocase for high affinity reception of non-folded preprotein clients. Such dynamics nexuses are likely universal and essential in multi-liganded protein machines.

biochemistry↗

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↗

bioRxiv2020

PyHDX: Derivation and visualization of protection factors from Hydrogen-Deuterium Exchange Mass Spectrometry at near residue resolution

Hydrogen Deuterium Exchange Mass Spectrometry is a powerful monitor of protein intrinsic dynamics, yet the interpretation, visualization and cross-comparison of HDX-MS datasets is challenging. Here we present PyHDX, an open-source python package and web server, that batch-extracts the universal quantity Gibbs free energy at residue level over multiple protein conditions and homologues. {Delta}G values relate to protein normal modes and together provide a universal measure of protein flexibility. AvailabilityPyHDX source code is released under the MIT license and can be accessed on GitHub.

bioinformatics↗

bioRxiv2019

ColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data

Single-molecule fluorescence microscopy studies of bacteria provide unique insights into the mechanisms of cellular processes and protein machineries in ways that are unrivalled by any other technique. With the cost of microscopes dropping and the availability of fully automated microscopes, the volume of microscopy data produced has increased tremendously. These developments have moved the bottleneck of throughput from image acquisition and sample preparation to data analysis. Furthermore, requirements for analysis procedures have become more stringent given the requirement of various journals to make data and analysis procedures available. To address this we have developed a new data analysis package for analysis of fluorescence microscopy data of rod-like cells. Our software ColiCoords structures microscopy data at the single-cell level and implements a coordinate system describing each cell. This allows for the transformation of Cartesian coordinates of both cellular images (e.g. from transmission light or fluorescence microscopy) and single-molecule localization microscopy (SMLM) data to cellular coordinates. Using this transformation, many cells can be combined to increase the statistical significance of fluorescence microscopy datasets of any kind. Coli-Coords is open source, implemented in the programming language Python, and is extensively documented. This allows for modifications for specific needs or to inspect and publish data analysis procedures. By providing a format that allows for easy sharing of code and associated data, we intend to promote open and reproducible research.\n\nThe source code and documentation can be found via the projects GitHub page.

bioinformatics↗