Biology subjects

Lichtinger, S. M.

Publications and source records attributed to Lichtinger, S. M..

3 recordsLinked to original sources

bioRxiv2024

The mechanism of mammalian proton-coupled peptide transporters

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1 and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

biophysics↗

bioRxiv2023

Tackling hysteresis in conformational sampling - how to be forgetful with MEMENTO

The structure of proteins has long been recognised to hold the key to understanding and engineering their function, and rapid advances in structural biology (and protein structure prediction) are now supplying researchers with an ever-increasing wealth of structural information. Most of the time, however, structures can only be determined in free energy minima, one at a time. While conformational flexibility may thus be inferred from static end-state structures, their interconversion mechanisms -- a central ambition of structural biology -- are often beyond the scope of direct experimentation. Given the dynamical nature of the processes in question, many studies have attempted to explore conformational transitions using molecular dynamics (MD). However, ensuring proper convergence and reversibility in the predicted transitions is extremely challenging. In particular, a commonly used technique to map out a path from a starting to a target conformation called targeted MD (tMD) can suffer from starting-state dependence (hysteresis) when combined with techniques such as umbrella sampling (US) to compute the free energy profile of a transition. Here, we study this problem in detail on conformational changes of increasing complexity. We also present a new, history-independent approach that we term "MEMENTO" (Morphing End states by Modelling Ensembles with iNdependent TOpologies) to generate paths that alleviate hysteresis in the construction of conformational free energy profiles. MEMENTO utilises template-based structure modelling to restore physically reasonable protein conformations based on coordinate interpolation (morphing) as an ensemble of plausible intermediates, from which a smooth path is picked. We compare tMD and MEMENTO on well-characterized test cases (the toy peptide deca-alanine and the enzyme adenylate kinase) before discussing its use in more complicated systems (the kinase P38 and the bacterial leucine transporter LeuT). Our work shows that for all but the simplest systems tMD paths should not in general be used to seed umbrella sampling or related techniques, unless the paths are validated by consistent results from biased runs in opposite directions. MEMENTO, on the other hand performs well as a flexible tool to generate intermediate structures for umbrella sampling. We also demonstrate that extended end-state sampling combined with MEMENTO can aid the discovery of collective variables on a case-by-case basis. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/525919v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a5de3corg.highwire.dtl.DTLVardef@1a17305org.highwire.dtl.DTLVardef@867721org.highwire.dtl.DTLVardef@1f73798_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Targeted modulation of protein liquid-liquid phase separation by evolution of amino-acid sequence

Rationally and efficiently modifying the amino-acid sequence of proteins to control their ability to undergo liquid-liquid phase separation (LLPS) on demand is not only highly desirable, but can also help to elucidate which protein features are important for LLPS. Here, we propose an innovative computational method that couples a genetic algorithm to a sequence-dependent coarse-grained protein model to evolve the amino-acid sequences of phase-separating intrinsically disordered protein regions (IDRs), and purposely enhance or inhibit their capacity to phase-separate. We apply it to the phase-separating IDRs of three naturally occurring proteins, namely FUS, hnRNPA1 and LAF1, as prototypes of regions that exist in cells and undergo homotypic LLPS driven by different types of intermolecular interaction. We find that the evolution of amino-acid sequences towards enhanced LLPS is driven in these three cases, among other factors, by an increase in the average size of the amino acids. However, the direction of change in the molecular driving forces that enhance LLPS (such as hydrophobicity, aromaticity and charge) depends on the initial amino-acid sequence: the critical temperature can be enhanced by increasing the frequency of hydrophobic and aromatic residues, by changing the charge patterning, or by a combination of both. Finally, we show that the evolution of amino-acid sequences to modulate LLPS is strongly coupled to the composition of the medium (e.g. the presence or absence of RNA), which may have significant implications for our understanding of phase separation within the many-component mixtures of biological systems.

biophysics↗