Biology subjects

Rees, D. C.

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

12 recordsLinked to original sources

bioRxiv2026

Structural basis of metalloid transport by the arsenite efflux pump ArsB

Bacteria resist toxic arsenite (AsIII) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of AsIII and antimonite (SbIII) reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an inward-facing conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that AsIII resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H+)-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pKa estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H+-coupling mechanism. Our findings provide structural insights into metalloid recognition and H+/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria.

biochemistry↗

bioRxiv2025

Nucleotide and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA

A common mechanism of arsenic detoxification in bacteria is arsenite (AsIII) efflux facilitated by the ArsAB pump that couples metalloid transport to ATP hydrolysis. The cytoplasmic ATPase component, ArsA, binds and hydrolyzes ATP and facilitates the transfer of AsIII to the integral membrane transporter, ArsB. The underlying molecular mechanism of AsIII efflux by ArsAB remains unclear. ArsA is a member of the Intradimeric Walker A (IWA) family of ATPases that undergo dramatic nucleotide-dependent conformational changes to facilitate their respective biological functions. Similar conformational transitions in ArsA have been postulated to drive AsIII binding and transport via ArsB but have not been demonstrated. Here, we report multiple structures of ArsA determined by single-particle cryogenic electron microscopy in an open MgADP-bound state, open MgATP-bound state, and a distinct closed MgATP-bound state liganded to AsIII. Using X-ray absorption spectroscopy, we confirmed that AsIII coordinates three conserved cysteines at the metalloid-binding site of the closed state in a three-coordinate fashion. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other members of the IWA family and provide the structural basis for allosteric activation of nucleotide hydrolysis by AsIII. This work enhances our understanding of how the ArsA catalytic cycle regulates metalloid efflux by ArsB.

biochemistry↗

bioRxiv2024

Postdoctoral Scholar Recruitment and Hiring Practices in STEM: A Pilot Study

Despite the importance of the postdoctoral position in the training of scientists for independent research careers, few studies have addressed recruiting and hiring of postdocs. We conducted a pilot study on postdoctoral hiring in the Division of Chemistry and Chemical Engineering at the California Institute of Technology to serve as a starting point to better understand postdoctoral recruiting and hiring processes. From this survey of both postdocs and faculty, together with the available literature, the picture emerges that the postdoc hiring process is more decentralized than either faculty hiring or graduate admissions. Postdoc positions are often filled through a passive process where the initial expression of interest from a prospective postdoc is through a "cold-call" contact to a prospective advisor. Individual faculty members are often responsible for developing and implementing their own outreach and recruitment plans and deciding who to hire into a postdoc position. The overall opacity of the processes and practices by which postdocs are identified, recruited, and hired make it difficult to pinpoint where interventions could be effective to ensure equitable hiring practices. Implementation of such practices is critical to training a diverse postdoc population and subsequently of the future STEM faculty recruited from this group.

scientific communication and education↗

bioRxiv2023

Modeling the Correlation between Z and B in an X-ray Crystal Structure Refinement

We have examined how the refined B-factor changes as a function of Z (the atomic number of a scatterer) at the sulfur site of the [4Fe:4S] cluster of the nitrogenase iron protein by refinement. A simple model is developed that quantitatively captures the observed relationship between Z and B, based on a Gaussian electron density distribution with a constant electron density at the position of the scatterer. From this analysis, the fractional changes in B and Z are found to be similar. The utility of B-factor refinement to potentially distinguish atom types reflects the Z dependence of X-ray atomic scattering factors; the weaker dependence of electron atomic scattering factors on Z implies that distinctions between refined values of B in an electron scattering structure will be less sensitive to the atomic identity of a scatterer than for the case with X-ray-diffraction. This behavior provides an example of the complementary information that can be extracted from different types of scattering studies.

biophysics↗

bioRxiv2022

Anaerobic single particle cryoEM of nitrogenase

The enzyme nitrogenase catalyzes the reduction of dinitrogen to ammonia during biological nitrogen fixation through a mechanism involving the ATP dependent interaction of two component proteins adopting multiple conformational states. To date, high resolution structural information has been provided by X-ray crystallography, which restricts the states that can be accessed to those that can be crystallized. Cryo-electron microscopy (cryoEM) presents a new opportunity for structural characterization of nitrogenase solution structures, and may yield new information on the mechanism of nitrogenase by revealing structures of transient or heterogeneous states. In this study, we present single particle cryoEM structures of the MoFe-nitrogenase endogenously isolated from Azotobacter vinelandii. To maintain the fully reduced cluster states of this oxygen sensitive protein, we prepared samples within an anaerobic chamber and employed specialized conditions to minimize partial disordering of the -subunit at the air-water interface during freezing. Under these conditions, cryoEM structures of the as-isolated MoFe-protein and stabilized MoFe-protein-Fe-protein ADP-AlF4-complex were generally found to closely resemble their corresponding X-ray crystallographic structures. The cryoEM structures did reveal disordering in regions of the MoFe-protein -subunit reminiscent of that observed previously for the {Delta}nifB MoFe-protein lacking the FeMo-cofactor, suggesting that this disorder may reflect functionally relevant dynamics, as well as the possibility of asymmetric binding of the Fe-protein to the MoFe-protein in solution. The methods presented here pave the way toward the capture and interrogation of turnover-relevant nitrogenase states by cryoEM.

biochemistry↗

bioRxiv2022

Selenocyanate Derived Se-Incorporation into the Nitrogenase Fe Protein Cluster

The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein independent substrate (CO2) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe4S4 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe4S4 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source. The observed chalcogenide exchange illustrates that this Fe4S4 cluster is capable of core substitution reactions under certain conditions, adding to the Fe proteins repertoire of unique properties.

biochemistry↗

bioRxiv2022

The erythrocyte membrane properties of beta thalassaemia heterozygotes and their consequences for Plasmodium falciparum invasion

Malaria parasites such as Plasmodium falciparum have exerted formidable selective pressures on the human genome. Of the human genetic variants associated with malaria protection, beta thalassaemia (a haemoglobinopathy) was the earliest to be associated with malaria prevalence. However, the malaria protective properties of beta thalassaemic erythrocytes remain unclear. Here we studied the mechanics and surface protein expression of beta thalassaemia heterozygous erythrocytes, measured their susceptibility to P. falciparum invasion, and calculated the energy required for merozoites to invade them. We found invasion-relevant differences in beta thalassaemic cells versus matched controls, specifically: elevated membrane tension, reduced bending modulus, and higher levels of expression of the major invasion receptor basigin. However, these differences acted in opposition to each other with respect to their likely impact on invasion, and overall we did not observe beta thalassaemic cells to have lower P. falciparum invasion efficiency for any of the strains tested.

microbiology↗

bioRxiv2021

Glutathione binding to the plant At Atm3 transporter and implications for the conformational coupling of ABC transporters

The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined structures of AtAtm3 in multiple conformational states. These structures not only provide a structural framework for defining the alternating access transport cycle, but also highlight an unappreciated feature of the glutathione binding site, namely the paucity of cysteine residues that could potentially form inhibitory mixed disulfides with glutathione. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 with bound GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures containing both nucleotide and transport substrate suggests that this ternary complex exists only transiently during the transport cycle.

biochemistry↗

bioRxiv2021

Fluorescence Activation Mechanism and Imaging of Drug Permeation with New Sensors for Smoking-Cessation Ligands

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ("iDrugSnFRs") for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

pharmacology and toxicology↗

bioRxiv2021

Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that MetQ is a lipoprotein

NmMetQ is a substrate binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To address the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the absence and presence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function/topology, playing a role in NmMetNI-mediated transport at the inner-membrane in addition to moonlighting functions on the bacterial surface.

biochemistry↗

bioRxiv2020

A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

neuroscience↗

bioRxiv2019

Crosslinking of nucleotide binding domains improves the coupling efficiency of an ABC transporter

ATP Binding Cassette (ABC) transporters often exhibit significant basal ATPase activity in the absence of transported substrates. To investigate the factors that contribute to this inefficient coupling of ATP hydrolysis to transport, we characterized the structures and functions of variants of the bacterial Atm1 homolog from Novosphingobium aromaticivorans (NaAtm1), including forms with disulfide crosslinks between the nucleotide binding domains. Unexpectedly, disulfide crosslinked variants of NaAtm1 reconstituted into proteoliposomes not only transported oxidized glutathione, but also exhibited more efficient coupling of ATP hydrolysis to GSSG transport than the native transporter. These observations suggest that enhanced conformational dynamics of reconstituted NaAtm1 may contribute to the inefficient use of ATP. Understanding the origins of this uncoupled ATPase activity, and reducing the impact through disulfide crosslinking or other protocols, will be critical for the detailed dissection of ABC transporter mechanism to assure that the ATP dependent steps are indeed relevant to substrate translocation.

biochemistry↗