FlhF regulates the number and configuration of periplasmic flagella in Borrelia burgdorferi.

The Lyme disease bacterium Borrelia burgdorferi has 7-11 periplasmic flagella (PF) that arise from the cell poles and extend toward the midcell as a flat-ribbon, which is distinct from other bacteria. FlhF, a signal recognition particle (SRP)-like GTPase, has been found to regulate the flagellar number and polarity; however, its role in B. burgdorferi remains unknown. B. burgdorferi has an FlhF homolog (BB0270). Structural and biochemical analyses show that BB0270 has a similar structure and enzymatic activity as its counterparts from other bacteria. Genetics and cryo-electron tomography studies reveal that deletion of BB0270 leads to mutant cells that have less PF (4 ± 2 PF per cell tip) and fail to form a flat-ribbon, indicative of a role of BB0270 in the control of PF number and configuration. Mechanistically, we demonstrate that BB0270 localizes at the cell poles and controls the number and position of PF via regulating the flagellar protein stability and the polar localization of the MS-ring protein FliF. Our study not only provides the detailed characterizations of BB0270 and its profound impacts on flagellar assembly, morphology and motility in B. burgdorferi, but also unveils mechanistic insights into how spirochetes control their unique flagellar patterns.

Detergent-free systems for structural studies of membrane proteins.

Membrane proteins play vital roles in living organisms, serving as targets for most currently prescribed drugs. Membrane protein structural biology aims to provide accurate structural information to understand their mechanisms of action. The advance of membrane protein structural biology has primarily relied on detergent-based methods over the past several decades. However, detergent-based approaches have significant drawbacks because detergents often damage the native protein-lipid interactions, which are often crucial for maintaining the natural structure and function of membrane proteins. Detergent-free methods recently have emerged as alternatives with a great promise, e.g. for high-resolution structure determinations of membrane proteins in their native cell membrane lipid environments. This minireview critically examines the current status of detergent-free methods by a comparative analysis of five groups of membrane protein structures determined using detergent-free and detergent-based methods. This analysis reveals that current detergent-free systems, such as the styrene-maleic acid lipid particles (SMALP), the diisobutyl maleic acid lipid particles (DIBMALP), and the cycloalkane-modified amphiphile polymer (CyclAPol) technologies are not better than detergent-based approaches in terms of maintenance of native cell membrane lipids on the transmembrane domain and high-resolution structure determination. However, another detergent-free technology, the native cell membrane nanoparticles (NCMN) system, demonstrated improved maintenance of native cell membrane lipids with the studied membrane proteins, and produced particles that were suitable for high-resolution structural analysis. The ongoing development of new membrane-active polymers and their optimization will facilitate the maturation of these new detergent-free systems.

Structure and activity of lipid bilayer within a membrane-protein transporter.

Membrane proteins function in native cell membranes, but extraction into isolated particles is needed for many biochemical and structural analyses. Commonly used detergent-extraction methods destroy naturally associated lipid bilayers. Here, we devised a detergent-free method for preparing cell-membrane nanoparticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å resolution. We discovered a remarkably well-organized lipid-bilayer structure associated with transmembrane domains of the AcrB trimer. This bilayer patch comprises 24 lipid molecules; inner leaflet chains are packed in a hexagonal array, whereas the outer leaflet has highly irregular but ordered packing. Protein side chains interact with both leaflets and participate in the hexagonal pattern. We suggest that the lipid bilayer supports and harmonizes peristaltic motions through AcrB trimers. In AcrB D407A, a putative proton-relay mutant, lipid bilayer buttresses protein interactions lost in crystal structures after detergent-solubilization. Our detergent-free system preserves lipid-protein interactions for visualization and should be broadly applicable.

Multi-crystal native SAD analysis at 6†keV.

Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in de novo structure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6 keV. Increased anomalous signals were obtained at 6 keV compared with 7 keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2 Šresolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0 Šresolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.

Kinetic, mutational, and structural analysis of malonate semialdehyde decarboxylase from Coryneform bacterium strain FG41: mechanistic implications for the decarboxylase and hydratase activities.

Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal ion-independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide and a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. In terms of pairwise sequence, MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) is 38% identical with the Pseudomonas enzyme, including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. To determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of the enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for Pp MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily.

Fabrication of membrane proteins in the form of native cell membrane nanoparticles using novel membrane active polymers.

Membrane proteins are a widespread class of bio-macromolecules responsible for numerous vital biological processes and serve as therapeutic targets for a vast array of contemporary medications. For membrane protein isolation and purification, detergents have historically been used. Despite this, detergents frequently result in protein instability. Consequently, their application was limited. Recent detergent-free approaches have been invented. Among these, styrene-maleic acid lipid particle (SMALP), diisobutylene-maleic acid lipid particle (DIBMALP), and native cell membrane nanoparticle (NCMN) systems are the most prevalent. The NCMN system intends to create a library of membrane-active polymers suitable for high-resolution structure determination of membrane protein. Design, synthesis, characterization, and comparative application evaluations of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x, and NCMNP21b-x, are presented in this article. Although each NCMN polymer can solubilize distinct model membrane proteins and retain native lipids in NCMN particles, only the NCMNP21b-x family produces lipid-protein particles with ideal buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. NCMNP21b-x polymers that generate high-quality NCMN particles are particularly desirable for membrane protein structural biology.

Characterization of Ca2+-ATPase, LMCA1, with native cell membrane nanoparticles system.

Ca2+-ATPases are membrane pumps that transport calcium ions across the cell membrane and are dependent on ATP. The mechanism of Listeria monocytogenes Ca2+-ATPase (LMCA1) in its native environment remains incompletely understood. LMCA1 has been investigated biochemically and biophysically with detergents in the past. This study characterizes LMCA1 using the detergent-free Native Cell Membrane Nanoparticles (NCMNP) system. As demonstrated by ATPase activity assays, the NCMNP7-25 polymer is compatible with a broad pH range and Ca2+ ions. This result suggests that NCMNP7-25 may have a wider array of applications in membrane protein research.

pH-tunable membrane-active polymers, NCMNP2a-x, and their potential membrane protein applications.

Accurate 3D structures of membrane proteins are essential for comprehending their mechanisms of action and designing specific ligands to modulate their activities. However, these structures are still uncommon due to the involvement of detergents in the sample preparation. Recently, membrane-active polymers have emerged as an alternative to detergents, but their incompatibility with low pH and divalent cations has hindered their efficacy. Herein, we describe the design, synthesis, characterization, and application of a new class of pH-tunable membrane-active polymers, NCMNP2a-x. The results demonstrated that NCMNP2a-x could be used for high-resolution single-particle cryo-EM structural analysis of AcrB in various pH conditions and can effectively solubilize BcTSPO with the function preserved. Molecular dynamic simulation is consistent with experimental data that shed great insights into the working mechanism of this class of polymers. These results demonstrated that NCMNP2a-x might have broad applications in membrane protein research.

Merging cultures and disciplines to create a drug discovery ecosystem at Virginia commonwealth university: Medicinal chemistry, structural biology, molecular and behavioral pharmacology and computational chemistry.

The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.

Analysis of the oligomeric state of mycobacterial membrane protein large 3 and its interaction with SQ109 with native cell membrane nanoparticles system.

Mycobacterial membrane protein large 3 (Mmpl3) as a trehalose monomycolate lipid transporter contributes to cell wall biosynthesis. Inhibition of Mmpl3 can suppress cell growth and lead to mycobacterial death. SQ109 is a hydrophobic inhibitor of Mmpl3. We have devised a detergent-free strategy to characterize the SQ109/Mmpl3 interaction using the Native Cell Membrane Nanoparticles (NCMN) system, a new method for extracting membrane proteins that better retains native lipids. The homogeneity of the Mmpl3 NCMN particles was confirmed with electron microscopy. The hydrophobic protein-ligand interaction analysis shown for Mmpl3 using the NCMN system may broadly apply to other membrane proteins.

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase: Implications for the catalytic and inactivation mechanisms.

The isomeric mixture of cis- and trans-1,3-dichloropropene constitutes the active component of a widely used nematocide known as Telone II®. The mixture is processed by various soil bacteria to acetaldehyde through the 1,3-dichloropropene catabolic pathway. The pathway relies on an isomer-specific hydrolytic dehalogenation reaction catalyzed by cis- or trans-3-chloroacrylic acid dehalogenase, known respectively as cis-CaaD and CaaD. Previous sequence analysis and crystallographic studies of the native and covalently modified enzymes identified Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114 as key binding and catalytic residues in cis-CaaD. Mutagenesis of these residues confirmed their importance to the dehalogenation reaction. Crystal structures of the native enzyme (2.01Å resolution) and the enzyme covalently modified at the Pro-1 nitrogen by 2-hydroxypropanoate (1.65Å resolution) are reported here. Both structures are at a resolution higher than previously reported (2.75Å and 2.1Å resolution, respectively). The conformation of the covalent adduct is strikingly different from that previously reported due to its interaction with a 7-residue loop (Thr-32 to Leu-38). The participation of another active site residue, Arg-117, in catalysis and inactivation was also examined. The implications of the combined findings for the mechanisms of catalysis and inactivation are discussed.

3D interaction homology: Hydropathic interaction environments of serine and cysteine are strikingly different and their roles adapt in membrane proteins.

Atomic-resolution protein structural models are prerequisites for many downstream activities like structure-function studies or structure-based drug discovery. Unfortunately, this data is often unavailable for some of the most interesting and therapeutically important proteins. Thus, computational tools for building native-like structural models from less-than-ideal experimental data are needed. To this end, interaction homology exploits the character, strength and loci of the sets of interactions that define a structure. Each residue type has its own limited set of backbone angle-dependent interaction motifs, as defined by their environments. In this work, we characterize the interactions of serine, cysteine and S-bridged cysteine in terms of 3D hydropathic environment maps. As a result, we explore several intriguing questions. Are the environments different between the isosteric serine and cysteine residues? Do some environments promote the formation of cystine S-S bonds? With the increasing availability of structural data for water-insoluble membrane proteins, are there environmental differences for these residues between soluble and membrane proteins? The environments surrounding serine and cysteine residues are dramatically different: serine residues are about 50% solvent exposed, while cysteines are only 10% exposed; the latter are more involved in hydrophobic interactions although there are backbone angle-dependent differences. Our analysis suggests that one driving force for -S-S- bond formation is a rather substantial increase in burial and hydrophobic interactions in cystines. Serine and cysteine become less and more, respectively, solvent-exposed in membrane proteins. 3D hydropathic environment maps are an evolving structure analysis tool showing promise as elements in a new protein structure prediction paradigm.

Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities.

Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from the heterogeneity of the polymers and nonspecific interactions between the polymers and the relatively large hydrophobic pockets within the transmembrane domain of YnaI. However, this limitation offers development opportunities for detergent-free technology for challenging membrane proteins.

A native cell membrane nanoparticles system allows for high-quality functional proteoliposome reconstitution.

Proteoliposomes mimic the cell membrane environment allowing for structural and functional membrane protein analyses as well as antigen presenting and drug delivery devices. To make proteoliposomes, purified functional membrane proteins are required. Detergents have traditionally been used for the first step in this process However, they can irreversibly denature or render membrane proteins unstable, and the necessary removal of detergents after reconstitution can decrease proteoliposome yields. The recently developed native cell membrane nanoparticles (NCMN) system has provided a variety of detergent-free alternatives for membrane protein preparation for structural biology research. Here we attempt to employ the MCMN system for the functional reconstitution of channels into proteoliposomes. NCMN polymers NCMNP1-1 and NCMNP7-1, members of a NCMN polymer library that have been successful in extraction and affinity purification of a number of intrinsic membrane proteins, were selected for the purification and subsequent reconstitution of three bacterial channels: KcsA and the mechanosensitive channels of large and small conductance (MscL and MscS). We found that channels in NCMN particles, which appeared to be remarkably stable when stored at 4 °C, can be reconstituted into bilayers by simply incubating with lipids. We show that the resulting proteoliposomes can be patched for electrophysiological studies or used for the generation of liposome-based nanodevices. In sum, the findings demonstrate that the NCMN system is a simple and robust membrane protein extraction and reconstitution approach for making high-quality functional proteoliposomes that could significantly impact membrane protein research and the development of nanodevices.

Structural Basis of Antibody Conformation and Stability Modulation by Framework Somatic Hypermutation.

Accumulation of somatic hypermutation (SHM) is the primary mechanism to enhance the binding affinity of antibodies to antigens in vivo. However, the structural basis of the effects of many SHMs remains elusive. Here, we integrated atomistic molecular dynamics (MD) simulation and data mining to build a high-throughput structural bioinformatics pipeline to study the effects of individual and combination SHMs on antibody conformation, flexibility, stability, and affinity. By applying this pipeline, we characterized a common mechanism of modulation of heavy-light pairing orientation by frequent SHMs at framework positions 39H, 91H, 38L, and 87L through disruption of a conserved hydrogen-bond network. Q39LH alone and in combination with light chain framework 4 (FWR4L) insertions further modulated the elbow angle between variable and constant domains of many antibodies, resulting in improved binding affinity for a subset of anti-HIV-1 antibodies. Q39LH also alleviated aggregation induced by FWR4L insertion, suggesting remote epistasis between these SHMs. Altogether, this study provides tools and insights for understanding antibody affinity maturation and for engineering functionally improved antibodies.

Be Cautious with Crystal Structures of Membrane Proteins or Complexes Prepared in Detergents.

Membrane proteins are an important class of macromolecules found in all living organisms and many of them serve as important drug targets. In order to understand their biological and biochemical functions and to exploit them for structure-based drug design, high-resolution and accurate structures of membrane proteins are needed, but are still rarely available, e.g., predominantly from X-ray crystallography, and more recently from single particle cryo-EM - an increasingly powerful tool for membrane protein structure determination. However, while protein-lipid interactions play crucial roles for the structural and functional integrity of membrane proteins, for historical reasons and due to technological limitations, until recently, the primary method for membrane protein crystallization has relied on detergents. Bicelle and lipid cubic phase (LCP) methods have also been used for membrane protein crystallization, but the first step requires detergent extraction of the protein from its native cell membrane. The resulting, crystal structures have been occasionally questioned, but such concerns were generally dismissed as accidents or ignored. However, even a hint of controversy indicates that methodological drawbacks in such structural research may exist. In the absence of caution, structures determined using these methods are often assumed to be correct, which has led to surprising hypotheses for their mechanisms of action. In this communication, several examples of structural studies on membrane proteins or complexes will be discussed: Resistance-Nodulation-Division (RND) family transporters, microbial rhodopsins, Tryptophan-rich Sensory Proteins (TSPO), and Energy-Coupling Factor (ECF) type ABC transporters. These analyses should focus the attention of membrane protein structural biologists on the potential problems in structure determination relying on detergent-based methods. Furthermore, careful examination of membrane proteins in their native cell environments by biochemical and biophysical techniques is warranted, and completely detergent-free systems for membrane protein research are crucially needed.

Crystal structure of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase.

Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Šresolution is reported. The crystals belonged to space group P3221, with unit-cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (Rfree = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Functional Characterization of Sex Pheromone Receptors in the Fall Armyworm (Spodoptera frugiperda).

Pheromone receptors (PRs) found in the antennae of male moths play a vital role in the recognition of sex pheromones released by females. The fall armyworm (FAW), Spodoptera frugiperda, is a notorious invasive pest, but its PRs have not been reported. In this report, six candidate PRs (SfruOR6, 11, 13, 16, 56 and 62) suggested by phylogenetic analysis were cloned, and their tissue-sex expression profiles were determined by quantitative real-time PCR (qPCR). All six genes except for SfruOR6 were highly and specifically expressed in the antennae, with SfruOR6, 13 and 62 being male-specific, while the other three (SfruOR11, 16 and 56) were male biased, suggesting their roles in sex pheromone perception. A functional analysis by the Xenopus oocyte system further demonstrated that SfruOR13 was highly sensitive to the major sex pheromone component Z9-14:OAc and the pheromone analog Z9,E12-14:OAc, but less sensitive to the minor pheromone component Z9-12:OAc; SfruOR16 responded weakly to pheromone component Z9-14:OAc, but strongly to pheromone analog Z9-14:OH; the other four candidate PRs did not respond to any of the four pheromone components and four pheromone analogs. This study contributes to clarifying the pheromone perception in the FAW, and provides potential gene targets for developing OR-based pest control techniques.