Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

Sperm chromatin-condensing protamine enhances SMYD5 thermal stability.

Studying thermal stability of proteins not only provides insight into protein structure but also is instrumental in identifying previously unknown interaction partners. We develop a machine learning strategy that combines orthogonal partial least squares regression and stability screening of Silver Bullets Bio library to identify biologically active molecules that enhance protein stability. This strategy proves effective in extracting the stability-enhancing molecules for SMYD5, a histone lysine methyltransferase that regulates chromosome integrity. Protamine, a histone substitute in chromatin condensation during spermatogenesis, is identified as the most influential molecule to enhance SMYD5 thermal stability. We find that the C-terminal poly-glutamic acid tract (poly-E) and a 30-residue insertion in MYND domain (M-insertion), which are unique to SMYD5, regulate the structural stability. However, protamine plays a dominant role in SMYD5 stability, and in the presence of protamine, the poly-E tract or M-insertion loses its ability to affect the stability. The stability-enhancing effect of protamine is SMYD5 specific, and for SMYD2, a closely related homolog, protamine exhibits opposite, destabilizing effects. We find that both SMYD5 and SMYD2 interact with protamine, where SMYD5 interaction is independent of the poly-E tract and M-insertion. Protamine not only helps provide insight into the structure-stability relationships of SMYD5, but also suggests a potential functional link of SMYD5 to spermatogenesis. SMYD5 is a ubiquitously expressed gene with the highest expression in testis, especially in the seminiferous ducts that contain germ cells. Thus, our study opens up avenues that could help delineate major mechanisms underlying chromatin dynamics during spermatogenesis.

Structural and Antiviral Studies of the Human Norovirus GII.4 Protease.

Norovirus is the leading cause of acute gastroenteritis worldwide with a yearly reported 700 million cases driving a $60 billion global socioeconomic burden. With no United States Food and Drug Administration approved therapeutics and the chance for severe chronic infection and life-threatening complications, researchers have identified the protease as a potential target. However, drug development has focused on the norovirus GI.1 strain despite its accounting for less than 5% of all outbreaks. Our lab aims to change focus for norovirus drug design from GI.1 to the highly infective GII.4, responsible for more than 50% of all outbreaks worldwide. With the first published crystal structure of the norovirus GII.4 protease, we have identified several significant differences in the structure and active site that have hindered development of a potent inhibitor targeting the norovirus GII.4 protease. With these new insights, we have begun designing compounds that demonstrate increased inhibition of the clinically most relevant norovirus GII.4 strain.

Structural basis of PDZ-mediated chemokine receptor CXCR2 scaffolding by guanine nucleotide exchange factor PDZ-RhoGEF.

The CXC chemokine receptor 2 (CXCR2) is a G protein coupled receptor mediating interleukin-8 chemotactic signaling and plays an important role in neutrophil mobility and tumor migration. However, efficient CXCR2 signaling requires PDZ domain-mediated scaffolding of signaling complexes at the plasma membrane and functional coupling of the signaling to specific downstream signaling pathways, in which only one PDZ protein has been characterized to interact with CXCR2. Here, we identified five novel CXCR2-binding PDZ-containing proteins, among which PDZ-RhoGEF is of particular interest because this PDZ and RGS-containing guanine nucleotide exchange factor (GEF) is also involved in cell signaling and mobility. To reveal the molecular basis of the interaction, we solved the crystal structure of PDZ-RhoGEF PDZ domain in complex with the CXCR2 C-terminal PDZ binding motif. The structure reveals that the PDZ-CXCR2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four CXCR2 residues contributing to specific interactions. Structural comparison of CXCR2-binding PDZ domains and PDZ-RhoGEF PDZ bound with different ligands reveals PDZ- and ligand-specific interactions that may underlie the ability of promiscuous CXCR2 binding by different PDZ domains and PDZ binding promiscuity. The structure also reveals an unexpected asymmetric disulfide bond-linked PDZ dimer that allows simultaneous parallel binding of CXCR2 to two PDZ domains. This study provides not only the structural basis for PDZ-mediated CXCR2-PDZ-RhoGEF interaction, but also a new mode of PDZ dimerization, which both could prove valuable in understanding signaling complex scaffolding in CXCR2 signaling and coupling to specific signaling pathways.

Increased activity of unlinked Zika virus NS2B/NS3 protease compared to linked Zika virus protease.

Zika virus (ZIKV) is a flavivirus spread by daytime-active Aedes spp. mosquitoes such as A. aegypti and A. albopictus. Previously thought to be a mild infection, the latest ZIKV outbreak in the Americas is causally associated with more severe symptoms as well as severe birth defects, such as microcephaly. Currently no vaccine or antiviral exists. However, recent progress has demonstrated the viral NS2B/NS3 protease may be a suitable target for the development of small-molecule antiviral agents. To better understand the ZIKV protease, we expressed, purified, and characterized unlinked and linked NS2B/NS3 protease corresponding to an isolate from the recent outbreak in Puerto Rico. Unlinked ZIKV protease is more active and binds substrate with greater affinity than linked ZIKV protease. Therefore, we propose that unlinked ZIKV protease be used when evaluating or designing ZIKV protease inhibitors. Additionally, potent inhibitors of related viral proteases, like West Nile Virus and Dengue virus, may serve as advanced starting points to identify and develop ZIKV protease inhibitors.

Structure and function of SET and MYND domain-containing proteins.

SET (Suppressor of variegation, Enhancer of Zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF1) domain-containing proteins (SMYD) have been found to methylate a variety of histone and non-histone targets which contribute to their various roles in cell regulation including chromatin remodeling, transcription, signal transduction, and cell cycle control. During early development, SMYD proteins are believed to act as an epigenetic regulator for myogenesis and cardiomyocyte differentiation as they are abundantly expressed in cardiac and skeletal muscle. SMYD proteins are also of therapeutic interest due to the growing list of carcinomas and cardiovascular diseases linked to SMYD overexpression or dysfunction making them a putative target for drug intervention. This review will examine the biological relevance and gather all of the current structural data of SMYD proteins.

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter.

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains.

The Nogo-B receptor (NgBR) is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs). Small Angle X-ray Scattering (SAXS) analysis reveals the radius of gyration (Rg) of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax) of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD) analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.

A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity.

Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.

Protein crystallization: Eluding the bottleneck of X-ray crystallography.

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins.

New open conformation of SMYD3 implicates conformational selection and allostery.

SMYD3 plays a key role in cancer cell viability, adhesion, migration and invasion. SMYD3 promotes formation of inducible regulatory T cells and is involved in reducing autoimmunity. However, the nearly "closed" substrate-binding site and poor in vitro H3K4 methyltransferase activity have obscured further understanding of this oncogenically related protein. Here we reveal that SMYD3 can adopt an "open" conformation using molecular dynamics simulation and small-angle X-ray scattering. This ligand-binding-capable open state is related to the crystal structure-like closed state by a striking clamshell-like inter-lobe dynamics. The two states are characterized by many distinct structural and dynamical differences and the conformational transition pathway is mediated by a reversible twisting motion of the C-terminal domain (CTD). The spontaneous transition from the closed to open states suggests two possible, mutually non-exclusive models for SMYD3 functional regulation and the conformational selection mechanism and allostery may regulate the catalytic or ligand binding competence of SMYD3. This study provides an immediate clue to the puzzling role of SMYD3 in epigenetic gene regulation.

Purification of Histone Lysine Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from Estrogen Receptor α.

Methylation of estrogen receptor α by the histone lysine methyltransferase SMYD2 regulates ERα chromatin recruitment and its target gene expression. This protocol describes SMYD2 purification and crystallization of SMYD2 in complex with an ERα peptide. Recombinant SMYD2 is overexpressed in Escherichia coli cells. After release from the cells by French Press, SMYD2 is purified to apparent homogeneity with multiple chromatography methods. Nickel affinity column purifies SMYD2 based on specific interaction of its 6×His tag with the bead-immobilized nickel ions. Desalting column is used for protein buffer exchange. Gel filtration column purifies SMYD2 based on molecular size. The entire purification process is monitored and analyzed by SDS-polyacrylamide gel electrophoresis. Crystallization of SMYD2 is performed with the hanging drop vapor diffusion method. Crystals of the SMYD2-ERα peptide complex are obtained by microseeding using seeding bead. This method can give rise to large size of crystals which are suitable for X-ray diffraction data collection. X-ray crystallographic study of the SMYD2-ERα complex can provide structural insight into posttranslational regulation of ERα signaling.

Molecular Dynamics Simulation Reveals Correlated Inter-Lobe Motion in Protein Lysine Methyltransferase SMYD2.

SMYD proteins are an exciting field of study as they are linked to many types of cancer-related pathways. Cardiac and skeletal muscle development and function also depend on SMYD proteins opening a possible avenue for cardiac-related treatment. Previous crystal structure studies have revealed that this special class of protein lysine methyltransferases have a bilobal structure, and an open-closed motion may regulate substrate specificity. Here we use the molecular dynamics simulation to investigate the still-poorly-understood SMYD2 dynamics. Cross-correlation analysis reveals that SMYD2 exhibits a negative correlated inter-lobe motion. Principle component analysis suggests that this correlated dynamic is contributed to by a twisting motion of the C-lobe with respect to the N-lobe and a clamshell-like motion between the lobes. Dynamical network analysis defines possible allosteric paths for the correlated dynamics. There are nine communities in the dynamical network with six in the N-lobe and three in the C-lobe, and the communication between the lobes is mediated by a lobe-bridging ß hairpin. This study provides insight into the dynamical nature of SMYD2 and could facilitate better understanding of SMYD2 substrate specificity.

Targeting the Root Cause of Cystic Fibrosis.

Cystic Fibrosis (CF) is a serious genetic condition caused by CF transmembrane conductance regulator (CFTR) mutation. CF patients have shortened lifespan due to airway obstruction, infection, and end-stage lung failure. However, recent development in CF therapy suggests a brighter future for CF patients. Targeting specific CFTR mutations aims to potentiate the channel gating activity of impaired CFTR and restore protein trafficking to the plasma membrane. Gene therapy introduces correct CFTR gene into the affected airway epithelium leading to the functional expression of CFTR in CF patients. This review will sum up the current status in CF-cause targeting therapy.

PDZ Structure and Implication in Selective Drug Design against Cystic Fibrosis.

PDZ domains play an essential role in a number of cellular processes by facilitating protein scaffolding and assembly of protein complexes. These domains consist of 80 to 90 amino acids and are found to recognize short C-terminal sequences of target proteins. Protein complex formation between PDZ target molecules can lead to a number of signaling and regulatory cascades that may either promote or inhibit the activation of certain proteins. It has been shown that the interaction of the PDZ domains of NHERF2 with LPA2 plays an inhibitory role on the cystic fibrosis transmembrane conductance regulator (CFTR) by promoting the assembly of a CFTR-NHERF2-LPA2 complex. CFTR regulates chloride ion transport across the epithelial plasma membrane, and individuals possessing CFTR mutations show decreased protein function and consequently, viscous mucus accumulation due to improper fluid transport. This type of ailment is termed cystic fibrosis. Thus, insight to the structure of PDZ domains and how they function to form macromolecular complexes could be therapeutically important in augmenting CFTR channel activity in cystic fibrosis patients. Here we review the PDZ domain family while dissecting their structure, function and implications in CFTR regulation and cystic fibrosis.