Bacterial membrane proteins make up ∼ 30% of the prokaryotic genome and play key roles in infection and virulence. Membrane protein chemistry has advanced in recent years, including purification strategies that mimic nativelike lipid environments, such as lipid nanoparticles, amphipols, and nanodiscs. The use of styrene maleic acid copolymers (SMALPs) to form a lipid nanoparticle has become increasingly common in membrane protein purification, especially for proteins which are not amenable to detergent extraction from the cellular membrane fraction. Yet, for some biochemical and biophysical methods, it is preferable to use detergent-solubilized protein. Here we show a general exchange screening method to transfer membrane proteins from lipid nanoparticles to detergent micelles while retaining protein fold, homogeneity, and function. Conditions were first optimized for copolymer dispersion and recovery into detergents, and analytical methods were employed to assess activity and quality of detergent-solubilized proteins. Thirteen protein targets were purified in copolymer based on a 16-polymer screen. This selection was followed by an eight-detergent screen in the presence of calcium and magnesium ions for optimal dissolution of the nanoparticle, producing detergent-stabilized protein. In all membrane proteins assessed, homogeneity and folding were retained from the initial purification in lipid nanoparticles through a detergent-exchange protocol. For membrane enzymes that have proven to be experimentally intractable when detergent solubilized, we were able to observe catalytic activity using the detergent-exchanged material. The use of this protocol to purify membrane proteins provides greater versatility for biochemical and kinetic characterization than was previously accessible.

Single-domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell-surface display. However, for membrane proteins, the targets are generally detergent-solubilized, and there remains a need for Nb development methods against membrane proteins in a native-like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as styrene-maleic acid to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to four membrane-resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane-associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways. The selected Nbs showed high affinity and selectivity towards their target proteins with KD apparent values ranging from 15 nM to 200 nM, depending on the Nb-protein conjugate. In accordance with their tight binding, various Nb-protein complexes were found to be stable to size-exclusion chromatography purification. The Nbs were also amenable to sortase-mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high-affinity Nb against membrane proteins in SMALP will facilitate their widespread application in cell biology and biomedical applications.

Human lectins are critical carbohydrate-binding proteins that recognize diverse glycoconjugates from microorganisms and can play a key role in host-microbe interactions. Despite their importance in immune recognition and microbe binding, the specific glycan ligands and functions of many human lectins remain poorly understood. Using previous proof-of-concept studies on selected lectins as the foundation for this work, we present ten additional glycan analysis probes (GAPs) from a diverse set of human soluble lectins, offering robust tools to investigate glycan-mediated interactions. We describe a protein engineering platform that enables scalable production of GAPs that maintain native-like conformations and oligomerization states, equipped with functional reporter tags for targeted glycan profiling. We demonstrate that the soluble GAP reagents can be used in various applications, including glycan array analysis, mucin-binding assays, tissue staining, and microbe binding in complex populations. These capabilities make GAPs valuable for dissecting interactions relevant to understanding host responses to microbes. The tools can also be used to probe differential microbial and mammalian glycan interactions, which are crucial for understanding the interactions of lectins in a physiological environment where both glycan types exist. GAPs have potential as diagnostic and prognostic tools for detecting glycan alterations in chronic diseases, microbial dysbiosis, and immune-related conditions.

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.

Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in the bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives.

Monotopic phosphoglycosyl transferases (monoPGTs) are an expansive superfamily of enzymes that catalyze the first membrane-committed step in the biosynthesis of bacterial glycoconjugates. MonoPGTs show a strong preference for their cognate nucleotide diphospho-sugar (NDP-sugar) substrates. However, despite extensive characterization of the monoPGT superfamily through previous development of a sequence similarity network comprising >38,000 nonredundant sequences, the connection between monoPGT sequence and NDP-sugar substrate specificity has remained elusive. In this work, we structurally characterize the C-terminus of a prototypic monoPGT for the first time and show that 19 C-terminal residues play a significant structural role in a subset of monoPGTs. This new structural information facilitated the identification of co-conserved sequence "fingerprints" that predict NDP-sugar substrate specificity for this subset of monoPGTs. A Hidden Markov model was generated that correctly assigned the substrate of previously unannotated monoPGTs. Together, these structural, sequence, and biochemical analyses have delivered new insight into the determinants guiding substrate specificity of monoPGTs and have provided a strategy for assigning the NDP-sugar substrate of a subset of enzymes in the superfamily that use UDP-di-N-acetyl bacillosamine. Moving forward, this approach may be applied to identify additional sequence motifs that serve as fingerprints for monoPGTs of differing UDP-sugar substrate specificity.

The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community.

Bacterial virulence and viability are strongly dependent on extracellular glycoconjugates including lipopolysaccharide (LPS), capsular polysaccharide (CPS), wall teichoic acid (WTA) and glycoproteins. While these glycoconjugates are diverse in structure and function, they share a common biosynthetic logic. For example, in Gram‐negative bacteria the biosynthesis of a variety of glycoconjugates are initiated on the cytoplasmic face of the bacterial inner membrane, with a phosphoglycosyl transferase (PGT) catalyzing the first membrane‐committed step of the pathway. PGTs transfer a phosphosugar from a nucleotide‐activated donor substrate onto a polyprenol phosphate such as undecaprenol phosphate (UndP). PGTs comprise two distinct superfamilies – polytopic, with multiple membrane spanning helices, and the more recently described monotopic, which contain an unusual re‐entrant membrane intercalating helix. To date, monotopic PGTs have only been observed in bacteria, and remain underexplored antimicrobial targets. In an effort to understand NDP‐sugar specificity and inform inhibitor design, a library of monotopic PGTs from human pathogens was expressed, purified, and subjected to structural and functional characterization. Both detergent and styrene maleic acid (SMA) copolymers were assessed for solubilization towards the purification and stabilization of these challenging protein targets. These efforts have led to purified samples of diverse monotopic PGTs for activity analyses using the UMP‐Glo ™ assay and crystallization screens, which have afforded reproduceable crystals for further investigation.Support or Funding InformationR01GM131627, F32GM134576

In humans, a collection of diverse proteins known as lectins are responsible for binding to glycan epitopes. Recognition of extracellular glycan epitopes represents a key facet of the innate immune response in eukaryotes. These epitopes can be used to distinguish cell types, self‐from non‐self, and perhaps even pathogenic from commensal bacteria. Understanding human lectin specificity can uncover mechanisms underlying innate immunity. Additionally, lectins represent attractive reagents for the development of glycan‐reading probes. Here, we demonstrate our efforts to develop and deploy a pipeline for recombinant expression, functionalization, and characterization of a subset of human lectins, including C‐type lectins, ficolins, jacalin‐type lectins, galectins, and intelectins. Target lectins can be efficiently expressed and purified, functionalized with either biotin or a fluorophore via sortase‐mediated ligation under flow, and screened for glycan binding. The success of this pipeline has been validated in preliminary studies showing that the recombinant lectins can engage with glycans from mammalian glycan arrays, bacterial glycan arrays, purified mucins, or human biological samples. These experiments highlight the potential of lectins as valuable molecular probes that fill a gap in the current molecular toolkit for glycan analysis.Support or Funding InformationNIH U01 CA231079‐01

Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.

Unsaturated fatty acids are vital constituents of cell membranes. Their relative abundance modulates membrane fluidity, providing organisms with mechanisms by which to maintain cell membrane homeostasis in response to changes in the external environment. In E. coli, unsaturation is introduced into fatty acids during - not after - de novo fatty acid biosynthesis by FabA, a dehydratase that not only catalyzes the dehydration of a key substrate in the pathway but also isomerizes the resulting product to yield a cis-unsaturated intermediate. A related dehydratase, FabZ, unlike FabA, has no isomerase activity, but complements FabA in fatty acid biosynthesis by processing a unique set of substrates. Though this presentation will emphasize the use of molecular dynamics (MD) simulations to delineate the origins of the distinct activities and substrate selectivities of these dehydratases, we hope it will also illustrate the value of using chemical, structural, and computational biology in concert to resolve the mechanisms by which biomolecules function.

Polyketide synthases (PKS) are a rich source of natural products of varied chemical composition and biological significance. Here, we report the characterization of an atypical dehydratase (DH) domain from the PKS pathway for gephyronic acid, an inhibitor of eukaryotic protein synthesis. Using a library of synthetic substrate mimics, the reaction course, stereospecificity, and tolerance to non-native substrates of GphF DH1 are probed via LC-MS analysis. Taken together, the studies establish GphF DH1 as a dual-function dehydratase/isomerase that installs an odd-to-even double bond and yields a product consistent with the isobutenyl terminus of gephyronic acid. The studies also reveal an unexpected C2 epimerase function in catalytic turnover with the native substrate. A 1.55-Å crystal structure of GphF DH1 guided mutagenesis experiments to elucidate the roles of key amino acids in the multistep DH1 catalysis, identifying critical functions for leucine and tyrosine side chains. The mutagenesis results were applied to add a secondary isomerase functionality to a nonisomerizing DH in the first successful gain-of-function engineering of a PKS DH. Our studies of GphF DH1 catalysis highlight the versatility of the DH active site and adaptation for a specific catalytic outcome with a specific substrate.

A sophisticated intracellular trafficking pathway in humans is used to tailor vitamin B-12 into its active cofactor forms, and to deliver it to two known B-12-dependent enzymes. Herein, we report an unexpected strategy for cellular retention of B-12, an essential and reactive cofactor. If methylmalonyl-CoA mutase is unavailable to accept the coenzyme B-12 product of adenosyltransferase, the latter catalyzes homolytic scission of the cobalt-carbon bond in an unconventional reversal of the nucleophilic displacement reaction that was used to make it. The resulting homolysis product binds more tightly to adenosyltransferase than does coenzyme B-12, facilitating cofactor retention. We have trapped, and characterized spectroscopically, an intermediate in which the cobalt-carbon bond is weakened prior to being broken. The physiological relevance of this sacrificial catalytic activity for cofactor retention is supported by the significantly lower coenzyme B-12 concentration in patients with dysfunctional methylmalonyl-CoA mutase but normal adenosyltransferase activity.

Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels, and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates, and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all-trans-trienoate-forming CurK-DH, whose kcat of 72 s-1 is more than 3 orders of magnitude greater than that of any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH DHs on the basis of incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ-DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology.

Natural and modified substrates coupled with LC-MS/MS analysis of products revealed the stereospecificity and stereoselectivity of a polyketide didomain.