Biochemistry

The biosynthetic machineries of ribosomally synthesized and post-translationally modified peptides (RiPPs) are often substrate tolerant. A remarkable example is the class II lanthipeptide synthetase ProcM, which naturally functions as a generalist enzyme that has not evolved to use a specific substrate during its evolutionary history. Although ProcM has been studied extensively, the sequence features associated with productive modification remain underexplored. In this study, we use the ultrahigh-throughput mRNA display technique to map the sequence compatibility of ProcM across a focused library. This approach expands the landscape of ProcM reactivity beyond native substrates and individually characterized variants. Machine learning (ML) is used as a tool to demonstrate that the selected dataset contains learnable signatures and classification architectures revealed a balanced accuracy of 0.73. This performance contrasts sharply with the near-perfect accuracy of specialized enzyme models as the sequence-fitness landscape of the generalist enzymes are characterized by class imbalance and limited by intrinsic dataset features. Our results provide a high-throughput view of ProcM reactivity and highlight differences with previous high-throughput studies on substrate selectivity of RiPP modification enzymes. Future studies will need to assess whether these differences are common when comparing generalist with specialist enzymes.

Sliding clamp proteins play central roles in DNA metabolism, including replication and repair. The ring-shaped E. coli beta clamp accommodates double-stranded DNA and serves as a platform for proteins involved in multiple DNA transactions. The inner pore of the beta clamp harbors a series of positively charged and polar residues that can bind to the negatively charged backbone of the DNA. These residues are arrayed so that they do not align with the charged phosphates of the DNA backbone. It is hypothesized that this arrangement of these residues provides for the movement of the clamp on DNA as it alternates which residues are bound to the DNA backbone. In this work, we mutated specific charged and polar residues that project into the inner pore of the beta clamp. The beta clamp variants are dimers and have similar thermal stability and in general a similar ability to complement a temperature sensitive strain for growth. One exception was beta-Q149A, which appeared as higher-order species on a native gel although its hydrogen-deuterium exchange pattern measured by mass spectrometry was overall similar to WT beta. These variants all had decreased binding to DNA after loading. Optical tweezers experiments were used to monitor loading on single DNA molecules and measure the rate of beta clamp sliding on DNA. Consistent with the hypothesized role of positively charged residues in the beta inner pore, mutation of one residue resulted in a faster rate of sliding on DNA.

Quorum sensing in Gram-positive bacteria commonly relies on posttranslationally modified peptide pheromones. In Bacillus subtilis, the prenyltransferase ComQ catalyzes tryptophan prenylation of the quorum-sensing peptide ComX, but the structural basis of this unique peptide modification has remained unclear. Here we identified a previously uncharacterized ComQ homolog, StheQ, and its cognate peptide substrate, StheX, from Sphaerobacter thermophilus and investigated their structural and functional relationship. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that StheQ catalyzes prenylation of the tryptophan residue located second from the C-terminus of StheX. Crystal structures of apo StheQ and its complexes with a farnesyl pyrophosphate analog revealed that StheQ adopts the all--helical fold of the trans-isoprenyl diphosphate synthase (IPPS) superfamily while possessing an active-site architecture adapted for peptide-based indole prenylation. The structures identified a single Mg2+-binding site associated with the first aspartic acid-rich motif and showed no evidence for metal coordination at the pseudo-second aspartic acid-rich motif. Site-directed mutagenesis, complex formation assays, and docking analyses identified a peptide-binding pocket adjacent to the active site and suggested that N215 contributes to productive positioning of the acceptor tryptophan. These findings establish the structural basis for peptide prenylation by a ComQ-family enzyme, providing insight into the evolution of peptide-based indole prenylation within the IPPS superfamily, and support the view that ComQ-family enzymes constitute a distinct functional branch specialized for peptide modification.

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

Oxalyl-coenzyme A (oxalyl-CoA) is a key intermediate in oxalate metabolism in plants, fungi and oxalate-degrading bacteria, but its limited availability has restricted biochemical investigations of oxalyl-CoA-dependent enzymes. Here, we describe a practical semisynthetic procedure for the preparation of oxalyl-CoA based on rapid oxalyl transfer from S-oxalyl p-thiocresol to coenzyme A. The reaction was monitored directly by 1H NMR spectroscopy, allowing optimisation of pD and reaction conditions. Following removal of thiocresol and purification by reversed-phase HPLC, oxalyl-CoA was obtained in 39% yield as determined by quantitative 1H NMR. The product was characterised by high-resolution electrospray mass spectrometry and comprehensive 1H, 13C and 31P NMR spectroscopy, confirming its structure unequivocally. During the study, the limited stability of oxalyl-CoA in aqueous solution was documented, leading to recommendations for its purification and storage. The semisynthetic protocol provides a convenient source of analytically pure oxalyl-CoA suitable for biochemical assays and supplies reference spectroscopic data for its unambiguous identification. The biological utility of the semisynthetic oxalyl-CoA was demonstrated by its application as an acyl donor substrate in assays of PnBAHD15, enabling quantitative kinetic characterisation of the enzyme and illustrating its suitability for biochemical studies of oxalyl-CoA-dependent enzymes.

BACH1 is a heme-regulated basic-leucine-zipper containing transcriptional repressor that binds its DNA recognition elements as a heterodimer with MAFK. Heme-binding is thought to be mediated by several Cys-Proline (CP) motifs and this results in dissociation of the heterodimer from DNA. The mechanism of heme-binding and heme-mediated DNA dissociation remains unresolved. We have used UV-visible spectroscopy, 2D-NMR and DNA-binding assays to explore both heme-binding and DNA dissociation of a minimal BACH1 construct containing 2 CP motifs (C492(CP5) and C646(CP6)) flanking the DNA-binding domain. We find that heme is able to bind to both CP motifs, but also to other non-CP cysteines and histidines in the construct. Using NMR spectroscopy, we identify a structured binding pocket in which heme interacts with both C646(CP6) and Cys621. However, DNA-binding assays show that C646(CP6) is not required for heme-mediated DNA dissociation of the BACH1:MAFK heterodimer. Using UV-visible spectroscopy we show that C492(CP5) also recruits heme with a second ligand, a conserved histidine, His559, in the BACH1 DNA-recognition helix. Mutation of C492(CP5) reduces but does not abolish heme-mediated dissociation from DNA. Our findings suggest a mechanism for heme-binding to BACH1 and heme-mediated dissociation from DNA.

Lanthipeptides are one of the largest classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). The coi biosynthetic gene cluster (BGC) from Streptomyces coelicolor A3(2) encodes a canonical class I lanthipeptide dehydratase (CoiB) and cyclase (CoiC), a bifunctional enzyme (CoiSA) with an O-methyltransferase (MT) and glutamyl lyase (GL) domain, and a protein of unknown function (CoiH). The product of the coi BGC was recently shown to impart anti-phage activity, but its structure is still unresolved. Previous work investigated the regioselectivity of the GL domains in CoiB and CoiSA and the stereochemistry of the cyclized precursor peptide, but the function of CoiH was not addressed. In this study, co-expression of the peptide CoiA1 with CoiBCSAH resulted in a +16 Da addition on the cyclized peptide compared to when CoiH was omitted. LC-MS/MS analysis indicated that this modification occurred in the first thioether ring. A combination of site-directed mutagenesis, comparison of linear and cyclized peptide substrates, hydrogen peroxide (H2O2) treatment, and collision-induced dissociation (CID) mass spectrometric analysis suggested that the sulfur atom in the first methyllanthionine was oxidized to a sulfoxide group by CoiH. This hypothesis was confirmed by NMR analysis. CoiH represents a previously uncharacterized oxygenase family catalyzing sulfoxide formation. Structure prediction tools suggest a novel enzyme fold without obvious metal or cofactor binding sites, raising the possibility that CoiH is a cofactor independent oxidation enzyme.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the {beta}7-{beta}8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the {beta}7-{beta}8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

This present work shows that E-hook fragments possess functional structure differences governed by electrostatic interactions and sequence composition. The acidic C-terminal tails of tubulin, known as E-hooks, play a central role in regulating interactions between microtubules and motor proteins, microtubule-associated proteins, and enzymatic modifiers. Despite their functional importance, the intrinsic structural properties of these peptide segments remain poorly characterized due to their intrinsically disordered nature. In this work, we present quantum-mechanically optimized structures of hexamer peptides derived from {beta}-tubulin E-hook sequences. Density functional theory calculations were used to optimize peptide geometries using progressively larger basis sets. From the optimized geometries we calculated theoretical Raman spectra, Ramachandran backbone dihedral distributions, and measured radii of gyration to resolve composition dependent structural tendencies. The combined Raman and conformational analyses provide a systematic computational approach for comparing simulated and experimental Raman spectra of tubulin E-hooks and other intrinsically disordered proteins and offer insight into how E-hooks contribute to the recognition mechanisms underlying the tubulin code.

We present a simple and efficient way to add cis dialdehydes, phosphoryl groups, or cis diols to the 3 prime end of oligonucleotides using periodate oxidized nucleotides (oNTPs) and terminal deoxynucleotidyl transferase (TdT). The 3 prime end cis dialdehyde-modified oligos are generated by incubating TdT with an oligo for several minutes followed by addition of a oNTP and incubated at 30 degrees C for 30 minutes to an hour. After allowing the addition of the cis dialdehydes, heating the reaction mixture at 90 to 95 degrees C for 10 minutes yields oligonucleotides with 3 prime phosphoryl groups. The 3 prime cis diol modified oligos are synthesized by starting with 3 prime cis diol nucleotides (HO-NTPs). The cis dialdehyde modified oligonucleotides and cis diols may then be used for a variety of investigations such as studying the interaction of proteins with the 3 prime end of DNA, and possibly RNA. As an example, we demonstrate the efficacy of using an oligonucleotide modified with oGMP at the 3 prime end as an affinity label for TdT and identified a peptide fragment that has been shown to contain two of three aspartate residues found to be in the TdT active site.

Mosaic penA alleles encoding highly mutated variants of penicillin-binding protein 2 (PBP2) are the principal determinants of ceftriaxone resistance in Neisseria gonorrhoeae. Resistance-associated mutations in PBP2 from the ceftriaxone-resistant strain H041 restrict formation of the inward conformation of the {beta}3-{beta}4 loop associated with efficient acylation, but how {beta}-lactam recognition is coupled to this conformational switch is unknown. Because the conserved active-site residue Tyr422 interacts with the R1 substituent of {beta}-lactams, we investigated its role in coupling ligand recognition and acylation activity. Mutation of Tyr422 to Ala lowered acylation rates by up to 120-fold for cefoperazone and piperacillin, whereas acylation rates of ceftriaxone increased 4-fold. Unexpectedly, the crystal structure of the Y422A mutant acylated by ceftriaxone revealed that the {beta}3-{beta}4 loop had adopted the inward, high-activity conformation, despite position 422 being spatially distant from the loop. Transformation experiments showed that cell viability requires a tyrosine at position 422, indicating the residue is essential for transpeptidase function. Together, these findings reveal an energetic coupling between an active-site residue in PBP2 and a conformational switch whose equilibrium is altered by resistance mutations. The previously observed higher activity of {beta}-lactams containing extended R1 groups is consistent with stronger interactions with Tyr422 that favor the conformational switch. Molecular modeling suggests that such groups enhance activity by mimicking the iso-Glu region of the pentapeptide substrate. Overall, we propose that access to the high-activity state of PBP2 where the {beta}3-{beta}4 loop is inward is regulated by interactions between Tyr422 and {beta}-lactam R1 groups, and that resistance mutations function by tilting the balance toward a lower activity state.

The enzymatic removal of sulfate groups regulates processes ranging from steroid metabolism to carbohydrate degradation. Most sulfatases belong to the S1 family, whose members use a co-translationally installed formylglycine residue to hydrolyse sulfate esters. Arylsulfamates are potent covalent inhibitors of aryl and steroid sulfatases, including the clinical steroid sulfatase inhibitor Irosustat, yet the structure and stability of the inhibited complex remain unresolved. Arylsulfamates and carbohydrate sulfamates do not covalently inhibit many S1 carbohydrate sulfatases despite conservation of their sulfate-binding sites and formylglycine residue. Using enzyme kinetics, X-ray crystallography, molecular dynamics simulations and density functional theory calculations, we define the basis of these contrasting behaviours. High-resolution structures of the Pseudomonas aeruginosa arylsulfatase PaAtsA treated with two arylsulfamates reveal a long-lived tetrahedral, O-linked -hydroxysulfamate adduct attached to formylglycine. Molecular simulations show that replacing sulfate with sulfamate disrupts the favourable Ca2+-oxyanion interaction and alters ligand binding geometry. The permissive hydrophobic binding site of PaAtsA accommodates this rearrangement while retaining a trajectory compatible with nucleophilic attack. By contrast, in the Bacteroides thetaiotaomicron carbohydrate sulfatase BT16363S-Gal, sulfate-to-sulfamate substitution weakens binding and displaces the sulfamate from a reactive pose near the catalytic nucleophile due to a restrictive active site with conserved sugar binding. These findings define the structure and persistence of the arylsulfamate-derived covalent intermediate and explain why sulfamate warheads are tolerated by aryl sulfatases but not carbohydrate sulfatases.

Selenocysteine (Sec), the 21st amino acid, is a rare non-canonical amino acid that represents an attractive target for protein engineering due to its desirable chemical properties such as high affinity for metals, strong nucleophilicity, and reversible covalent bond formation. To bypass the natural constraints on Sec placement within proteins, several strategies have been developed to rewire the native translational machinery to enable site-specific incorporation. However, these usually abolish the quality control mechanism that excludes the serine-charged selenocysteinyl-tRNA (Ser-tRNASec), the immediate biosynthetic precursor, from translation resulting in heterogenous protein species. This challenge is confounded by a lack of genetic tools to accurately report the selenylation state of the tRNA pool as most are blind to competing process of Ser incorporation, which can only be observed using analytical methods. To resolve this issue, we have developed a new fluorescent reporter, Selenocysteine Adjusted Ratiometric Chromophore (SeARCh), which exhibits two distinct spectral outputs dependent on the incorporation of either Ser (red) or Sec (green). Using SeARCh, we define several factors which influence the observed Sec:Ser ratio and construct a new hybrid biosynthetic pathway with improved performance, achieving 90% Sec incorporation. Furthermore, SeARCh displays unusually complex mass spectra due to the isotope distribution of selenium and heterogenous nature of the protein in solution and we report specific methods to account for this behaviour and precisely quantify the rare Ser-containing species found at high Sec incorporation efficiencies. Our findings suggest that the equilibrium between selenoprotein and tRNASec expression levels is a key driver of incorporation efficiency and implies a process that is broadly biosynthetically constrained. Collectively these tools represent a significant advance in the metrology of selenocysteine biosynthesis and incorporation and can be used to inform and standardize future engineering efforts.

UvrD-family Superfamily 1A helicases are processive ATP-dependent motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD-family monomers translocate along single stranded (ss) DNA with 3-to-5 directionality but must be activated by dimerization to become helicases in the absence of force or accessory factors. Mycobacterium tuberculosis (Mtb) UvrD1 helicase forms dimers via a disulfide bond between native cysteines in the 2B sub-domains of each monomer. E. coli UvrD forms non-covalent dimers using the same 2B domain interface as in Mtb UvrD1. Using both ensemble and single DNA molecule approaches we examined an E. coli UvrD variant (R421C), which forms covalent dimers with constitutive helicase activity. For the first time this has enabled us to compare the ssDNA translocation and helicase activities of covalent dimers and monomers. Crosslinked UvrD dimers exhibit much higher ssDNA translocation processivities than monomers, although with similar translocation rates. Crosslinked UvrD dimers also show highly processive DNA unwinding of thousands of base pairs, much higher than non-crosslinked UvrD dimers, while monomers show no DNA unwinding activity. DNA unwinding rates of crosslinked UvrD dimers are only [~]20% slower than ssDNA translocation rates, indicating they are "active" helicases that directly facilitate duplex destabilization.

The rotary motor F1-ATPase has been extensively studied as a model molecular machine, yet rational engineering of its catalytic activity remains challenging because ATP hydrolysis is regulated by long-range intersubunit allostery and large conformational transitions. Here, we developed a homolog-guided engineering strategy to increase the maximum rotation rate of the thermophilic Bacillus PS3 F1-ATPase (TF1). Candidate mutation sites were first identified by comparing TF1 with the homologous enzymes bovine mitochondrial F1 (bMF1) and Paracoccus denitrificans F1 (PdF1), both of which exhibit higher maximum rotation rates than TF1. Systematic exploration of these sites identified four activity-enhancing hotspots, followed by focused hotspot exploration and machine-learning-assisted prioritization of combinatorial mutants. The best mutant, TF1({beta}Y313L/{beta}E332S), exhibited a 1.8-fold higher maximum rotation rate than TF1(WT) while retaining its functional thermostability. Interestingly, activity-enhancing substitutions were not limited to the residues conserved in both bMF1 and PdF1, indicating that the bMF1-PdF1 consensus substitutions effectively identify activity-enhancing hotspots rather than uniquely defining the optimal amino acid. Machine-learning-assisted exploration efficiently prioritized highly active mutants, although the predictive performance was limited by the relatively small training dataset and epistatic interactions among mutations. Kinetic and structural comparisons further provided mechanistic insights into the enhanced catalytic activity of the engineered mutant. Together, these results establish a practical strategy for engineering complex molecular motors by combining homolog-guided hotspot identification with focused hotspot exploration.

Poly(ethylene terephthalate) (PET) is one of the most widely produced plastics, and enzymatic depolymerization offers a promising route to closed-loop recycling under mild conditions. However, most known bacterial PET hydrolases belong to a conserved canonical-fold cutinase family, leaving much of alpha/beta-hydrolase diversity unexplored. Here, we mapped bacterial cutinase sequence space by combining bioinformatics-guided sequence selection with high-throughput secretion screening in Bacillus subtilis. A library of 1,120 genes encoding 954 unique bacterial cutinases, spanning canonical- and minimal-fold families, was screened for activity on Impranil DLN and semicrystalline PET. We identified 156 secreted cutinases with polyester activity, broadly distributed across sequence space, but only ten showed detectable PET hydrolysis, all from the canonical-fold family. These PET hydrolases were active at 40-50{degrees}C, preferred alkaline pH, and showed moderate thermostability. Our results demonstrate that PET activity is rare among bacterial cutinases and provide a scalable workflow for discovering diverse enzyme starting points.

NicA2 is a flavin-bound amine dehydrogenase from Pseudomonas putida S16 that converts nicotine to the pharmacologically inactive N-methylmyosmine. In animal models of nicotine addiction, injection of NicA2 can decrease nicotine-seeking behavior 10-fold. Accordingly, NicA2-related enzymes have been investigated as smoking-cessation therapeutics. However, efficient catalysis by NicA2 in Pseudomonas putida relies on electron transfer to CycN, a cytochrome c, and not directly to O2. Impractically high amounts of NicA2 are thus necessary to achieve a pharmacological effect in the absence of CycN. Directed evolution has improved the ambient-O2 value of kcat from 0.007 s-1 to 1 s-1 for NicA2, but further improvements have been challenging. Here, we identify a strain of Peribacillus frigoritolerans NIC8 which encodes two flavin amine oxidoreductases, Ncox and Pnox. In the presence of oxygen, Ncox and Pnox act on nicotine and pseudooxynicotine respectively with apparent kcat values of 7.7 s-1 and 3.9 s-1. Transient kinetics establishes bimolecular rate constants of 51100 M-1s-1 and 81000 M-1s-1 for the half-reactions between Ncox and O2 and between Pnox and O2 respectively, consistent with Ncox and Pnox being bona-fide oxidases. Transcriptomics shows enhanced expression of Ncox and Pnox under nicotine-dependent growth as well as supporting the identification of downstream enzymes. Phylogenetic analysis suggests that Ncox and Pnox arose out of repurposing of homologous enzymes found in Bacillus species. The enzymes we describe may be useful for the development of nicotine addiction therapeutics and for bioconversion of nicotine in waste streams.

Cholesterol is a key component of eukaryotic cell membranes, promoting membrane stability and modulating the function of many membrane proteins, including ion channels. In our previous work using purified human voltage-gated proton channel proteins, we showed that cholesterol inhibits the hHv1 channel by altering the conformational dynamics of its S4 segment, the key element that senses membrane voltage to control proton permeation. In the present work, we examined the effects of cholesterol analogs and potential sites in the hHv1 channel mediating cholesterol inhibition using site-directed mutagenesis and docking simulations. Our results showed that desmosterol, the immediate precursor of cholesterol, markedly attenuates cholesterol inhibition. Using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we showed that desmosterol attenuates cholesterol inhibition by promoting the intermediate and open state conformations of the S4 segment. Moreover, we identified multiple residues in the hHv1 channel that are critical for cholesterol inhibition, including Y141A in the S2 segment, which reduces cholesterol inhibition by nearly 3-fold. Our smFRET results showed that the Y141A mutation promotes the intermediate conformation in the S4 segment, which underlies the attenuation of cholesterol inhibition. Consistently, docking simulations also revealed multiple residues spanning the transmembrane domain, rather than clustered within a single localized pocket. Our work identified the key molecular determinant in the hHv1 channel that mediates cholesterol inhibition and also provided a mechanism linking the conversion between demosterol and cholesterol by DHCR24 to pH homeostasis in many cells, such as phagocytes, cardiomyocytes, neurons and microglial cells.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.