Biology subjects

Hunt, J. F.

Publications and source records attributed to Hunt, J. F..

8 recordsLinked to original sources

bioRxiv2023

Cryo-EM studies of the four E. coli paralogs establish ABCF proteins as master plumbers of the peptidyl-transferase center of the ribosome

The genomes of most mesophilic organisms encode multiple ATP-Binding Cassette F (ABCF) proteins. EttA, one of four E. coli paralogs, regulates synthesis of the first peptide bond on the ribosome dependent on ATP/ADP ratio, while Antibiotic Resistance factors (AREs), paralogs in other organisms, both regulate and directly mediate resistance to ribosome-targeted antibiotics. However, the physiological functions remain unclear for most paralogs, and the mechanism-of-action has yet to be rigorously established for any paralog. We herein present single particle cryogenic electron microscopy structures of ribosome complexes of all four E. coli ABCF paralogs (EttA, Uup, YbiT, and YheS), which, together with previously determined ARE structures, show that ABCFs control the binding geometry of the tRNA in the peptidyl-tRNA-binding (P) site on the ribosome. They modulate the position of its acceptor stem relative to the peptidyl transferase center (PTC) in a manner that can either promote (EttA and Uup) or disrupt (YbiT, YheS, and the AREs) proper catalytic geometry. The YbiT/70S reconstructions include a conformation with no density for ribosomal protein bL33, and structural analyses support the exchange of this sub-stoichiometric ribosomal protein being functionally related to conformational changes in YbiT controlled by sequence variations in the strongly non-canonical Signature Sequence in its first ABC domain. Our studies establish general structural/enzymological principles by which the ATPase activity of ABCF proteins controls translation elongation coupled to modulation of conformation and stereochemistry in the catalytic core of the ribosome.

biophysics↗

bioRxiv2023

Comparative genetic, biochemical, and biophysical analyses of the four E. coli ABCF paralogs support distinct functions related to mRNA translation

Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four Escherichia coli K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A {Delta}uup knockout, like {Delta}ettA, exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither {Delta}ybiT nor {Delta}yheS exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on in vitro translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ2) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNAVal in the P site. However, EQ2-Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ2-YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ2-EttA and EQ2-YbiT fully inhibit in vitro translation of an mRNA encoding luciferase, while EQ2-Uup and EQ2-YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ2-Uup or EQ2-YheS, while EQ2-YbiT inhibits synthesis of both peptide bonds and EQ2-EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four E. coli ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.

biochemistry↗

bioRxiv2023

Systematic enhancement of protein crystallization efficiency by bulk lysine-to-arginine (KR) substitution

Structural genomics consortia established that protein crystallization is the primary obstacle to structure determination using x-ray crystallography. We previously demonstrated that crystallization propensity is systematically related to primary sequence, and we subsequently performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization and that these substitutions consistently enhance crystallization propensity. This approach rapidly produced a 1.9 [A] crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from bulk-KR-substituted domains show the engineered arginine residues frequently make high-quality hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve protein crystallization.

biophysics↗

bioRxiv2022

Mechanistic implications of the interaction of the soluble substrate-binding protein with a type II ABC importer

ATP-Binding Cassette (ABC) Transporters employ homologous ATPase domains to drive transmembrane transport of diverse substrates ranging from small molecules to large polymers. Bacterial ABC importers require an extramembranous substrate binding protein (SBP) to deliver the transport substrate to the extracellular side of the transporter complex. Previous studies suggest significant differences in the transport mechanisms of type I vs. type II bacterial ABC importers, which contain unrelated transmembrane domains. We herein use ensemble fluorescence resonance energy transfer (FRET) experiments to characterize the kinetics of SBP interaction in the E. coli BtuCD-F complex, a canonical type II ABC importer that transports vitamin B12. We demonstrate that, in the absence of B12, BtuF (the SBP) forms a locked (kinetically hyper-stable) complex with nanodisc-reconstituted BtuCD that can only be dissociated by ATP hydrolysis, which represents a futile reaction cycle. Notably, no type I importer has been observed to form an equivalent locked complex. We also show that either ATP or vitamin B12 binding substantially slows formation of the locked BtuCD-F complex, which will limit the occurrence of futile hydrolysis under physiological conditions. Mutagenesis experiments demonstrate that efficient locking requires concerted interaction of BtuCD with residues on both sides of the B12 binding pocket in BtuF. Combined with the kinetic inhibition of locking by ATP binding, these observations imply that the transition state for the locking reaction involves a global alteration in the conformation of BtuCD that extends from its BtuF binding site in the periplasm to its ATP-binding sites on the opposite side of the membrane in the cytoplasm. These observations suggest that locking, which seals the extracellular B12 entry site of the transporter, may help push B12 through the transporter and directly contribute to the transport mechanism in type II ABC importers.

biophysics↗

bioRxiv2022

Realtime observation of ATP-driven single B12 molecule translocationthrough BtuCD-F

ATP-Binding Cassette (ABC) Transporters use ATP binding and hydrolysis to power transmembrane transport of chemically diverse substrates. Current knowledge of their mechanism comes primarily from static structures of stable intermediates along the transport cycle. Recently, single-molecule fluorescence resonance energy transfer (smFRET) measurements have generated insight into the functional dynamics of transmembrane transporters, but studies to date lack direct information on the physical movement of the transport substrate. Here, we report development of an smFRET system that exploits fluorescence quenching by vitamin B12 to track its location in real time during ATP-driven transport by nanodisc-reconstituted E. coli BtuCD-F, an extensively studied type II ABC importer. Our data demonstrate that transmembrane translocation of B12 is driven by two sequential high-energy conformational changes that are inaccessible to standard structural methods because they are inherently transient. The first moves B12 from the periplasm into the transmembrane domain of the transporter; notably, this reaction is driven by hydrolysis of a single ATP molecule, in contrast to the mechanism established for several other ABC Transporter families in which ATP-binding drives the mechanochemical power-stroke prior to hydrolysis. The second mediates B12 release on the opposite side of the transporter, and it is driven by formation of a hyper-stable complex between BtuCD and BtuF. Hydrolysis of a second single ATP molecule is then required to dissociate BtuCD from the BtuF substrate-binding protein to enable it to bind B12 and initiate another round of transport. Our experiments have visualized substrate translocation in real-time at a single-molecule level and provided unprecedented information on the mechanism and dynamics of a paradigmatic transmembrane transport process.

biophysics↗

bioRxiv2022

Mechanism of dual pharmacological correction and potentiation of human CFTR

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 [A] non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis. SummaryCryo-EM global conformational ensemble reconstruction has been used to characterize the mechanism-of-action of a breakthrough pharmaceutical that corrects fatal protein-folding and channel-gating defects in the human cystic fibrosis transmembrane conductance regulator (CFTR).

biophysics↗

bioRxiv2022

Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5-carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions supports the notion that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters. Significance StatementTransporter proteins use energy to move molecular materials into and out of cells. To be efficient, the transporter motions responsible for moving the molecules must be tightly choreographed to avoid wasting energy without transporting anything. By measuring the motions and kinetics of a prototypical transporter (VcINDY) at the single-molecule level, this study finds the first evidence that transporters like VcINDY achieve efficient transport by coordinating constantly dynamic, "elevator-type" motions while sitting in the cellular membrane. The efficiency of these surprisingly dynamic transporters is then revealed by thermodynamic modeling, which explains the molecular basis behind how highly cooperative, substrate binding reactions may have evolved as the optimal strategy for maximizing transporter efficiency.

biophysics↗

bioRxiv2021

YhcB (DUF1043), a novel cell division protein conserved across gamma-proteobacteria

YhcB, an uncharacterized protein conserved across gamma-proteobacteria, is composed predominantly of a single Domain of Unknown Function (DUF 1043) with an N-terminal transmembrane -helix. Here, we show that E. coli YhcB is a conditionally essential protein that interacts with the proteins of the cell divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). We found 7 interactions of YhcB that are conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we identified several point mutations that abolished interactions of YhcB with FtsI and RodZ. The yhcB knock-out strain does not grow at 45{degrees}C and is hypersensitive to cell-wall acting antibiotics even in stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septa development. The 2.8 [A] crystal structure for the cytosolic domain from Haemophilus ducreyi YhcB shows a unique tetrameric -helical coiled-coil structure that combines parallel and anti-parallel coiled-coil intersubunit interactions. This structure is likely to organize interprotein oligomeric interactions on the inner surface of the cytoplasmic membrane, possibly involved in regulation of cell division and/or envelope biogenesis/integrity in proteobacteria. In summary, YhcB is a conserved and conditionally essential protein that is predicted to play a role in cell division and consequently or in addition affects envelope biogenesis. ImportanceOnly 0.8 % of the protein annotations in the UniProt are based on experimental evidence and thus, functional characterization of unknown proteins remains a rate-limiting step in molecular biology. Herein, the functional properties of YhcB (DUF1043) were investigated using an integrated approach combining X-ray crystallography with genetics and molecular biology. YhcB is a conserved protein that appears to be needed for the transition from exponential to stationary growth and is involved in cell division and/or envelope biogenesis/integrity. This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

microbiology↗