Biology subjects

Cafiso, D. S.

Publications and source records attributed to Cafiso, D. S..

6 recordsLinked to original sources

bioRxiv2021

Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport

Outer membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse EPR measurements were made in intact cells on the Escherichia coli vitamin B12 transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm towards the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, where TonB binding alone is sufficient to produce allosteric rearrangements in the transporter. They also demonstrate the importance of studying outer membrane proteins in their native environment.

biophysics↗

bioRxiv2020

Conserved Arginine Residues in Synaptotagmin 1 Regulate Fusion Pore Expansion Through Membrane Contact

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

neuroscience↗

bioRxiv2019

Partial metal ion saturation of C2 domains primes Syt1-membrane interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein that acts as a Ca2+ sensor of neurotransmitter release. How the Ca2+-sensing function of Syt1 is coupled to its interactions with anionic membranes and synaptic fusion machinery is not well understood. Here, we investigated the dynamics and membrane-binding properties of Syt1 under conditions where its highest affinity Ca2+ sites, which are thought to drive the initial membrane recruitment, are selectively populated by divalent metal ions. To create such protein states for the Ca2+-sensing C2 domains of Syt1, we exploited the unique chemistry of Pb2+, a xenobiotic metal ion that is isostructural and isofunctional to Ca2+. NMR experiments revealed that binding of a single metal ion results in the loss of conformational plasticity of the C2 domain loop regions that are involved in both coordinating metal ions and membrane interactions. In the C2A domain, a single metal ion is sufficient to drive its weak association with PtdSer-containing membranes; in C2B, it enhances the interactions with the signaling lipid PtdIns(4,5)P2. In full-length Syt1, both C2 domains associate with PtdSer-containing membranes, with the depth of insertion modulated by the occupancy of the metal ion sites. Our data suggest that Syt1 adopts a shallow membrane-bound state upon initial recruitment of its C2 domains to the membranes. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, \"prime\" Syt1 for binding a full complement of metal ions required for activation of protein function.

biochemistry↗

bioRxiv2019

Disulfide chaperone knock-outs enable in-vivo double spin-labeling of an outer-membrane transporter

Recent advances in the application of EPR spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer-membrane proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli outer-membrane vitamin B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated outer membranes or reconstituted systems, and the ability to label and perform EPR in cells is expected to be applicable to a range of other bacterial outer-membrane proteins.\n\nStatement of SignificanceEPR spectroscopy is an important method to characterize the structure and dynamics of membrane proteins, and recent efforts demonstrate that pulse EPR can be used to examine the extracellular surface of outer membrane proteins in live bacteria. In the present work, we show that pairs of cysteine residues in the Escherichia coli vitamin B12 transporter, BtuB, cannot be spin-labeled in wild-type strains, but can be labeled with the use of certain null mutants in the periplasmic disulfide bond formation, Dsb, system. These mutants also facilitate efficient spin-labeling of cysteines located on the periplasmic surface of BtuB. Distance measurements using pulse EPR provide evidence that the behavior of BtuB is different in the bacterial cell than it is in purified systems.

biophysics↗

bioRxiv2019

Hybrid refinement of heterogeneous conformational ensembles using spectroscopic data

Multi-structured biomolecular systems play crucial roles in a wide variety of cellular processes but have resisted traditional methods of structure determination which often resolve only a few low-energy states. High-resolution structure determination using experimental methods that yield distributional data remains extremely difficult, especially when the underlying conformational ensembles are quite heterogeneous. We have therefore developed a method to integrate sparse, multi-multimodal spectroscopic data to obtain high-resolution estimates of conformational ensembles. We have tested our method by incorporating double electron-electron resonance data on the SNARE protein syntaxin-1a into biased molecular dynamics simulations. We find that our method substantially outperforms existing state-of-the-art methods in capturing syntaxins open/closed conformational equilibrium and further yields new conformational states that are both consistent with experimental data and may help in understanding syntaxins function. Our improved methods for refining heterogeneous conformational ensembles from spectroscopic data will greatly accelerate the structural understanding of such systems.

biophysics↗

bioRxiv2019

Phosphatidylinositol 4,5 bisphosphate controls the cis and trans interactions of synaptotagmin 1

Synaptotagmin 1 acts as the Ca2+-sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here we describe measurements on full-length membrane reconstituted synaptotagmin 1 using site-directed spin labeling where we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate (PIP2), even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+-binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS containing bilayers and will bind in a cis configuration to membranes containing PS even if a PIP2 membrane is presented in trans. The data are consistent with a bridging activity for Syt1 where the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

neuroscience↗