Biology subjects

Marx, D. C.

Publications and source records attributed to Marx, D. C..

6 recordsLinked to original sources

bioRxiv2025

Structural Diversity of Metabotropic Glutamate Receptor/Beta-Arrestin Coupling

Beta-arrestins ({beta}-arrs) are cytosolic proteins which mediate G protein-coupled receptor (GPCR) desensitization, endocytosis, and signaling. Despite the widespread physiological roles of {beta}-arr coupling, the molecular basis of GPCR/{beta}-arr interaction has been studied primarily in monomeric family A GPCRs. Here we take an integrative biophysical and structural approach to uncover molecular diversity in {beta}-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. We find, using a new single molecule pulldown assay, that mGluRs couple to {beta}-arrs with a 2:1 or 2:2 stoichiometry via a combination of "tail" and "core" interactions. Using single molecule FRET analysis, we also find that {beta}-arr1 stabilizes active conformations of mGluR8. Cryo-EM structures of mGluR8 alone or with either G proteins or {beta}-arr1 reveal transducer-specific mGluR8 active states and, in combination with molecular dynamics simulations, define the positioning of mGluR8-bound {beta}-arr1, supporting a steric mechanism of mGluR desensitization involving interactions with both subunits and the lipid bilayer. Finally, combinatorial mutagenesis enables the identification of a landscape of homo- and hetero-dimeric mGluR/{beta}-arr complexes, including mGluR/{beta}-arr1/{beta}-arr2 megacomplexes, providing a framework for family C GPCR/{beta}-arr coupling and expanding the known range of GPCR/transducer coupling modes.

biophysics↗

bioRxiv2023

Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals a novel mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily. Significance StatementG protein-coupled receptors (GPCRs) allow cells to sense and respond to their environment and constitute the largest class of targets for approved therapeutic drugs. Temporally precise GPCR signaling is achieved by coupling the binding of extracellular ligands to the binding of intracellular signal transducers (e.g. heterotrimeric G proteins) and regulators (e.g. {beta}-arrestins). The C-terminal domains (CTDs) of GPCRs are targets of various post-translational modifications and play a critical role in transducer and regulator recruitment. Here we report novel interactions of the CTDs of two GPCRs of the metabotropic glutamate receptor family with cellular membranes. These interactions serve to regulate CTD accessibility and thus, mGluR coupling to transducers and regulators. We propose that dynamic CTD-membrane interaction constitutes a general mechanism for regulating GPCR function.

biophysics↗

bioRxiv2022

FkpA Enhances Membrane Protein Folding using an Extensive Interaction Surface

Outer membrane protein (OMP) biogenesis in gram-negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones bind unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress uOMP aggregation, facilitate diffusion across the periplasm, and enhance OMP folding. FkpA primarily responds to heat-shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function, we monitored the folding of a cognate client uOmpA171 and found that FkpA increases the folded uOmpA171population but also slows the folding rate, dual functions distinct from the other periplasmic chaperones. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to directly influence the uOmpA171folding trajectory. We determine the binding affinity between FkpA and uOmpA171 by globally fitting sedimentation velocity titrations and found it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggestive of an extensive binding interface. Initial characterizations of the complex using photo-crosslinking indicates that the binding interface spans the inner surfaces of the entire FkpA molecule. In contrast to prior findings, folding and binding experiments performed using subdomain constructs of FkpA demonstrate that the full-length chaperone is required for full activity. Together these results support that FkpA has a distinct and direct effect on uOMP folding and that it achieves this by utilizing an extensive chaperone-client interface. SignificanceThe periplasmic chaperone network is required for the survival and virulence of gram-negative bacteria. Here we find that the chaperone FkpA enhances outer membrane protein folding and tightly binds its clients with an extensive interaction interface. This modified holdase function of FkpA distinguishes it from other periplasmic chaperones and complements their functions to ensure robust outer membrane biogenesis.

biophysics↗

bioRxiv2020

De novo design of transmembrane beta-barrels

The ability of naturally occurring transmembrane {beta}-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a "hypothesis, design and test" approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the {beta}-turns and {beta}-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history. One sentence summarySuccess in de novo design of transmembrane {beta}-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

synthetic biology↗

bioRxiv2020

Water Controls Membrane Protein Stability

Through the insertion of nonpolar side chains into the bilayer, the hydrophobic effect has long been accepted as a driving force for membrane protein folding. However, how the changing chemical composition of the bilayer affects the magnitude side chain transfer free energies [Formula] has historically not been well understood. A particularly challenging region for experimental interrogation is the bilayer interfacial region that is characterized by a steep polarity gradient. In this study we have determined the [Formula] for nonpolar side chains as a function of bilayer position using a combination of experiment and simulation. We discovered an empirical correlation between the surface area of nonpolar side chain, the transfer free energies, and the local water concentration in the membrane that allows for [Formula] to be accurately estimated at any location in the bilayer. Using these water-to-bilayer [Formula] values, we calculated the interface-to-bilayer transfer free energy [Formula]. We find that the [Formula] are similar to the "biological", translocon-based transfer free energies, indicating that the translocon energetically mimics the bilayer interface. Together these findings can be applied to increase the accuracy of computational workflows used to identify and design membrane proteins, as well as bring greater insight into our understanding of how disease-causing mutations affect membrane protein folding and function.

biophysics↗

bioRxiv2019

SurA is a "Groove-y" Chaperone That Expands Unfolded Outer Membrane Proteins

The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network both due to its genetic interaction with the {beta}-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well understood. Here we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA*uOMP complex. We validated key structural features of the SurA*uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network. Significance StatementOuter membrane proteins play critical roles in bacterial physiology and increasingly are exploited as antibiotic targets. SurA is the most important chaperone in the OMP biogenesis network and is thought to initiate their folding through an interaction with the BAM complex. We observe an unprecedented expansion of unfolded outer membrane proteins when bound to SurA. This expansion suggests a potential mechanism by which SurA can deliver uOMPs to the BAM complex. In addition, this study highlights the use of an integrative/hybrid structural biology approach and emerging methods to map highly heterogeneous structural ensembles such as that of an unfolded protein bound to a chaperone.

biophysics↗