Atomic view of the HIV-1 matrix lattice; implications on virus assembly and envelope incorporation.

During the late phase of HIV type 1 (HIV-1) infection cycle, the virally encoded Gag polyproteins are targeted to the inner leaflet of the plasma membrane (PM) for assembly, formation of immature particles, and virus release. Gag binding to the PM is mediated by interactions of the N-terminally myristoylated matrix (myrMA) domain with phosphatidylinositol 4,5-bisphosphate. Formation of a myrMA lattice on the PM is an obligatory step for the assembly of immature HIV-1 particles and envelope (Env) incorporation. Atomic details of the myrMA lattice and how it mediates Env incorporation are lacking. Herein, we present the X-ray structure of myrMA at 2.15 Å. The myrMA lattice is arranged as a hexamer of trimers with a central hole, thought to accommodate the C-terminal tail of Env to promote incorporation into virions. The trimer­trimer interactions in the lattice are mediated by the N-terminal loop of one myrMA molecule and α-helices I­II, as well as the 310 helix of a myrMA molecule from an adjacent trimer. We provide evidence that substitution of MA residues Leu13 and Leu31, previously shown to have adverse effects on Env incorporation, induced a conformational change in myrMA, which may destabilize the trimer­trimer interactions within the lattice. We also show that PI(4,5)P2 is capable of binding to alternating sites on MA, consistent with an MA­membrane binding mechanism during assembly of the immature particle and upon maturation. Altogether, these findings advance our understanding of a key mechanism in HIV-1 particle assembly.

Novel pneumococcal capsule type 33E results from the inactivation of glycosyltransferase WciE in vaccine type 33F.

The polysaccharide (PS) capsule is essential for immune evasion and virulence of Streptococcus pneumoniae. Existing pneumococcal vaccines are designed to elicit anticapsule antibodies; however, the effectiveness of these vaccines is being challenged by the emergence of new capsule types or variants. Herein, we characterize a newly discovered capsule type, 33E, that appears to have repeatedly emerged from vaccine type 33F via an inactivation mutation in the capsule glycosyltransferase gene, wciE. Structural analysis demonstrated that 33E and 33F share an identical repeat unit backbone [→5)-ß-D-Galf2Ac-(1→3)-ß-D-Galp-(1→3)-α-D-Galp-(1→3)-ß-D-Galf-(1→3)-ß-D-Glcp-(1→], except that a galactose (α-D-Galp) branch is present in 33F but not in 33E. Though the two capsule types were indistinguishable using conventional typing methods, the monoclonal antibody Hyp33FM1 selectively bound 33F but not 33E pneumococci. Further, we confirmed that wciE encodes a glycosyltransferase that catalyzes the addition of the branching α-D-Galp and that its inactivation in 33F strains results in the expression of the 33E capsule type. Though 33F and 33E share a structural and antigenic similarity, our pilot study suggested that immunization with a 23-valent pneumococcal PS vaccine containing 33F PS did not significantly elicit cross-opsonic antibodies to 33E. New conjugate vaccines that target capsule type 33F may not necessarily protect against 33E. Therefore, studies of new conjugate vaccines require knowledge of the newly identified capsule type 33E and reliable pneumococcal typing methods capable of distinguishing it from 33F.

Discovery and Characterization of Pneumococcal Serogroup 36 Capsule Subtypes, Serotypes 36A and 36B.

Streptococcus pneumoniae can produce a wide breadth of antigenically diverse capsule types, a fact that poses a looming threat to the success of vaccines that target pneumococcal polysaccharide (PS) capsule. Yet, many pneumococcal capsule types remain undiscovered and/or uncharacterized. Prior sequence analysis of pneumococcal capsule synthesis (cps) loci suggested the existence of capsule subtypes among isolates identified as "serotype 36" according to conventional capsule typing methods. We discovered these subtypes represent two antigenically similar but distinguishable pneumococcal capsule serotypes, 36A and 36B. Biochemical analysis of their capsule PS structure reveals that both have the shared repeat unit backbone [→5)-α-d-Galf-(1→1)-d-Rib-ol-(5→P→6)-ß-d-ManpNAc-(1→4)-ß-d-Glcp-(1→] with two branching structures. Both serotypes have a ß-d-Galp branch to Ribitol. Serotypes 36A and 36B differ by the presence of a α-d-Glcp-(1→3)-ß-d-ManpNAc or α-d-Galp-(1→3)-ß-d-ManpNAc branch, respectively. Comparison of the phylogenetically distant serogroup 9 and 36 cps loci, which all encode this distinguishing glycosidic bond, revealed that the incorporation of Glcp (in types 9N and 36A) versus Galp (in types 9A, 9V, 9L, and 36B) is associated with the identity of four amino acids in the cps-encoded glycosyltransferase WcjA. Identifying functional determinants of cps-encoded enzymes and their impact on capsule PS structure is key to improving the resolution and reliability of sequencing-based capsule typing methods and discovering novel capsule variants indistinguishable by conventional serotyping methods.

Structural characterization of HIV-1 matrix mutants implicated in envelope incorporation.

During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation. Conversely, Env incorporation can be rescued by a compensatory mutation in the MA trimer interface (Q63R). The impact of these MA mutations on the structure and trimerization properties of MA is not known. In this study, we employed NMR spectroscopy, X-ray crystallography, and sedimentation techniques to characterize the structure and trimerization properties of HIV-1 MA A45E, Q63R, T70R, and L75G mutant proteins. NMR data revealed that these point mutations did not alter the overall structure and folding of MA but caused minor structural perturbations in the trimer interface. Analytical ultracentrifugation data indicated that mutations had a minimal effect on the MA monomer-trimer equilibrium. The high-resolution X-ray structure of the unmyristoylated MA Q63R protein revealed hydrogen bonding between the side chains of adjacent Arg-63 and Ser-67 on neighboring MA molecules, providing the first structural evidence for an additional intermolecular interaction in the trimer interface. These findings advance our knowledge of the interplay of MA trimerization and Env incorporation into HIV-1 particles.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

Pneumococci Can Become Virulent by Acquiring a New Capsule From Oral Streptococci.

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.

Structural and biophysical characterizations of HIV-1 matrix trimer binding to lipid nanodiscs shed light on virus assembly.

During the late phase of the HIV-1 replication cycle, the viral Gag polyproteins are targeted to the plasma membrane for assembly. The Gag-membrane interaction is mediated by binding of Gag's N-terminal myristoylated matrix (MA) domain to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The viral envelope (Env) glycoprotein is then recruited to the assembly sites and incorporated into budding particles. Evidence suggests that Env incorporation is mediated by interactions between Gag's MA domain and the cytoplasmic tail of the gp41 subunit of Env (gp41CT). MA trimerization appears to be an obligatory step for this interaction. Insufficient production of a recombinant MA trimer and unavailability of a biologically relevant membrane system have been barriers to detailed structural and biophysical characterization of the putative MA-gp41CT-membrane interactions. Here, we engineered a stable recombinant HIV-1 MA trimer construct by fusing a foldon domain (FD) of phage T4 fibritin to the MA C terminus. Results from NMR experiments confirmed that the FD attachment does not adversely alter the MA structure. Employing hydrogen-deuterium exchange MS, we identified an MA-MA interface in the MA trimer that is implicated in Gag assembly and Env incorporation. Utilizing lipid nanodiscs as a membrane mimetic, we show that the MA trimer binds to membranes 30-fold tighter than does the MA monomer and that incorporation of PI(4,5)P2 and phosphatidylserine enhances the binding of MA to nanodiscs. These findings advance our understanding of a fundamental mechanism in HIV-1 assembly and provide a template for investigating the interaction of MA with gp41CT.

The tuberculosis necrotizing toxin is an NAD+ and NADP+ glycohydrolase with distinct enzymatic properties.

Upon host infection, Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) into the cytosol of infected macrophages, leading to host cell death by necroptosis. TNT hydrolyzes NAD+ in the absence of any exogenous cofactor, thus classifying it as a ß-NAD+ glycohydrolase. However, TNT lacks sequence similarity with other NAD+ hydrolyzing enzymes and lacks the essential motifs involved in NAD+ binding and hydrolysis by these enzymes. In this study, we used NMR to examine the enzymatic activity of TNT and found that TNT hydrolyzes NADP+ as fast as NAD+ but does not cleave the corresponding reduced dinucleotides. This activity of TNT was not inhibited by ADP-ribose or nicotinamide, indicating low affinity of TNT for these reaction products. A selection assay for nontoxic TNT variants in Escherichia coli identified four of six residues in the predicted NAD+-binding pocket and four glycine residues that form a cradle directly below the NAD+-binding site, a conserved feature in the TNT protein family. Site-directed mutagenesis of residues near the predicted NAD+-binding site revealed that Phe727, Arg757, and Arg780 are essential for NAD+ hydrolysis by TNT. These results identify the NAD+-binding site of TNT. Our findings also show that TNT is an NAD+ glycohydrolase with properties distinct from those of other bacterial glycohydrolases. Because many of these residues are conserved within the TNT family, our findings provide insights into understanding the function of the >300 TNT homologs.

The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery.

The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly.

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2 We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.

The Interplay between Calmodulin and Membrane Interactions with the Pleckstrin Homology Domain of Akt.

The Akt protein, a serine/threonine kinase, plays important roles in cell survival, apoptosis, and oncogenes. Akt is translocated to the plasma membrane for activation. Akt-membrane binding is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). It has been shown that Akt activation in breast cancer cells is modulated by calmodulin (CaM). However, the molecular mechanism of the interplay between CaM and membrane binding is not established. Here, we employed nuclear magnetic resonance (NMR) and biochemical and biophysical techniques to characterize how PI(3,4,5)P3, CaM, and membrane mimetics (nanodisc) bind to Akt(PHD). We show that PI(3,4,5)P3 binding to Akt(PHD) displaces the C-terminal lobe of CaM but not the weakly binding N-terminal lobe. However, binding of a PI(3,4,5)P3-embedded membrane nanodisc to Akt(PHD) with a 103-fold tighter affinity than PI(3,4,5)P3 is able to completely displace CaM. We also show that Akt(PHD) binds to both layers of the nanodisc, indicating proper incorporation of PI(3,4,5)P3 on the nanodisc surface. No detectable binding has been observed between Akt(PHD) and PI(3,4,5)P3-free nanodiscs, demonstrating that PI(3,4,5)P3 is required for membrane binding, CaM displacement, and Akt activation. Using pancreatic cancer cells, we demonstrate that inhibition of Akt-CaM binding attenuated Akt activation. Our findings support a model by which CaM binds to Akt to facilitate its translocation to the membrane. Elucidation of the molecular details of the interplay between membrane and CaM binding to Akt may help in the development of potential targets to control the pathophysiological processes of cell survival.

Position of O-Acetylation within the Capsular Repeat Unit Impacts the Biological Properties of Pneumococcal Serotypes 33A and 33F.

Streptococcus pneumoniae (pneumococcus) produces many capsule types that differ in their abilities to evade host immune recognition. To explain these serotype-dependent protective capacities, many studies have investigated capsular thickness or the interaction of the capsule with complement proteins, but the effects of small chemical modifications of the capsule on its function have not been studied. One small chemical modification found frequently among pneumococcal capsules is O-acetylation. Pneumococcal serotype 33A has two membrane-bound O-acetyltransferase genes, wciG and wcjE A 33A wcjE-deficient variant, 33F, occurs naturally and is increasing in prevalence in the wake of widespread conjugate vaccine use, but no wciG-deficient variants have been reported. To study the biological consequence of the loss of O-acetylation, we created wciG-deficient variants in both serotypes 33A and 33F, which we named 33X1 (ΔwciG) and 33X2 (ΔwciG ΔwcjE). Serotypes 33X1 and 33X2 express novel capsule types based on serological and biochemical analyses. We found that loss of WcjE-mediated O-acetylation appears not to affect cell wall shielding, since serotypes 33A and 33F exhibit comparable nonspecific opsonophagocytic killing, biofilm production, and adhesion to nasopharyngeal cells, though serotype 33F survived short-term drying better than serotype 33A. Loss of WciG-mediated O-acetylation in serotypes 33X1 and 33X2, however, resulted in a phenotype resembling that of nonencapsulated strains: increased cell wall accessibility, increased nonspecific opsonophagocytic killing, enhanced biofilm formation, and increased adhesion to nasopharyngeal cells. We conclude that WciG-mediated, but not WcjE-mediated, O-acetylation is important for producing protective capsules in 33A and that small chemical changes to the capsule can drastically affect its biological properties.

Discovery of Novel Pneumococcal Serotype 35D, a Natural WciG-Deficient Variant of Serotype 35B.

Pneumococcus (Streptococcus pneumoniae) remains a significant cause of morbidity and mortality, especially among those at the extremes of age. Its capsular polysaccharide is essential for systemic virulence. Over 90 serologically distinct pneumococcal capsular polysaccharides (serotypes) are recognized, but they are unequal in prevalence. Because antibodies against the capsule are protective, polysaccharide conjugate vaccines, which are constructed against the most prevalent serotypes, have caused great reductions in pneumococcal disease caused by these serotypes. In response, however, the relative prevalences of serotypes have shifted. Certain previously rare serotypes, such as serotype 35B, are increasing in prevalence. Serotype 35B is thus a likely future vaccine candidate, but due to their previous rarity, serotype 35B strains have not been scrutinized for underlying heterogeneity. We studied putative serotype 35B clinical isolates to assess the uniformity of their serological reactions. While most isolates exhibited the accepted serology of serotype 35B, one isolate failed to bind to critical serotyping reagents. We determined that the genetic basis for this aberrant serology was the presence of inactivating mutations in the O-acetyltransferase gene wciG Complementation studies in a wciG deletion strain verified that the mutant WciG was nonfunctional, and the serology of the mutant could be restored through complementation with a construct encoding a functional WciG. Nuclear magnetic resonance studies confirmed that the capsule of the WciG-deficient isolate lacked O-acetylation but was otherwise identical to serotype 35B. As this isolate expresses a unique serology with unique biochemistry and a stable genetic basis, we named its novel capsule serotype 35D.

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt.

The translocation of Akt, a serine/threonine kinase, to the plasma membrane is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon EGF stimulation of breast cancer cells, CaM co-localizes with Akt at the plasma membrane to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed NMR, biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PHD of Akt (dissociation constant = 100 nm). The interaction between CaM and Akt(PHD) is enthalpically driven, and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for binding. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P3 binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P3 upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.

Pneumococcus with the "6E" cps Locus Produces Serotype 6B Capsular Polysaccharide.

Genetic studies of serogroup 6 isolates ofStreptococcus pneumoniaeidentified putative serotype 6E. Although its capsular polysaccharide structure has not been elucidated, putative serotype 6E is described in an increasing number of studies as a potentially new serotype. We show here that SPEC6B, which is widely used as a target strain for serotype 6B opsonophagocytosis assays, has the genetic features of the putative serotype 6E but produces capsular polysaccharide identical to 6B capsular polysaccharide as determined by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR). Thus, putative serotype 6E is a mere genetic variant of serotype 6B. Also, SPEC6B is appropriate as a target strain for serotype 6B opsonophagocytosis assays. This example illustrates the difficulties of assigning new bacterial serotypes based on genetic findings alone.

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers.

Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P(2). There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P(2) binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2'-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1'-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1'-acyl chains of PI(4,5)P(2) and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.

Solution structure of calmodulin bound to the binding domain of the HIV-1 matrix protein.

Subcellular distribution of calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM co-localizes and interacts with the HIV-1 Gag protein in the cytosol of infected cells. Although it has been shown that binding of Gag to CaM is mediated by the matrix (MA) domain, the structural details of this interaction are not known. We have recently shown that binding of CaM to MA induces a conformational change that triggers myristate exposure, and that the CaM-binding domain of MA is confined to a region spanning residues 8-43 (MA-(8-43)). Here, we present the NMR structure of CaM bound to MA-(8-43). Our data revealed that MA-(8-43), which contains a novel CaM-binding motif, binds to CaM in an antiparallel mode with the N-terminal helix (α1) anchored to the CaM C-terminal lobe, and the C-terminal helix (α2) of MA-(8-43) bound to the N-terminal lobe of CaM. The CaM protein preserves a semiextended conformation. Binding of MA-(8-43) to CaM is mediated by numerous hydrophobic interactions and stabilized by favorable electrostatic contacts. Our structural data are consistent with the findings that CaM induces unfolding of the MA protein to have access to helices α1 and α2. It is noteworthy that several MA residues involved in CaM binding have been previously implicated in membrane binding, envelope incorporation, and particle production. The present findings may ultimately help in identification of the functional role of CaM in HIV-1 replication.

Structural and biophysical characterization of the interactions between the death domain of Fas receptor and calmodulin.

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 µM and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.

Discovery of Streptococcus pneumoniae serotype 6 variants with glycosyltransferases synthesizing two differing repeating units.

Streptococcus pneumoniae is a persistent, opportunistic commensal of the human nasopharynx and is the leading cause of community-acquired pneumonia. It expresses an anti-phagocytic capsular polysaccharide (PS). Genetic variation of the capsular PS synthesis (cps) locus is the molecular basis for structural and antigenic heterogeneity of capsule types (serotypes). Serogroup 6 has four known members (6A-6D) with distinct serologic properties, homologous cps loci, and structurally similar PSs. cps of serotypes 6A/6B have wciNα, encoding α-1,3-galactosyltransferase, whereas serotypes 6C/6D have wciNß encoding α-1,3-glucosyltransferase. Two atypical serogroup 6 isolates (named 6X11 and 6X12) have been discovered recently in Germany. Flow cytometric studies using monoclonal antibodies show that 6X11 has serologic properties of 6B/6D, whereas 6X12 has 6A/6C. NMR studies of their capsular PSs revealed that 6X11 and 6X12 have two different repeating units with a distribution of ~40:60 6B:6D and 75:25 6A:6C PS, respectively. Sequencing of the wciNα gene in 6X12 and 6X11 revealed single and double nucleotide substitutions, respectively, resulting in the amino acid changes A150T and D38N. Substitution of alanine with threonine at position 150 in a 6A strain was associated with hybrid serologic and chemical profiles like 6X12. The hybrid serotypes represented by 6X12 and 6X11 strains are now named serotypes 6F and 6G. Single amino acid changes in cps genes encoding glycosyltransferases can alter substrate specificities, permit biosynthesis of heterogeneous capsule repeating units, and result in new hybrid capsule types that may differ in their interaction with the immune system of the host.

Spectrum of pneumococcal serotype 11A variants results from incomplete loss of capsule O-acetylation.

Streptococcus pneumoniae is a significant bacterial pathogen that expresses >90 capsule serotypes. Conventional serotyping methods assume that each serotype is a genetically and antigenically distinct entity; however, recent investigations have revealed pneumococcal isolates that cannot be unambiguously serotyped because they share the properties of more than one serotype. Here, we employed a novel serotyping method and NMR spectroscopy to examine clinical isolates sharing properties of serotypes 11A and 11E. These ambiguous clinical isolates were provisionally named 11A variant (11Av) isolates. Serotype 11A pneumococci characteristically express capsule ß-galactose-6-O-acetylation (ßGal6OAc) mediated by the capsule synthesis gene wcjE, while 11E strains contain loss-of-function mutations in wcjE and completely lack the expression of ßGal6OAc. Although 11Av isolates also contained mutated wcjE alleles, 11Av clinical isolates were composed of antigenically homogeneous bacteria expressing reduced amounts of 11A-specific capsule antigen. NMR data confirmed reduced but detectable amounts of ßGal6OAc on 11Av capsule polysaccharide. Furthermore, the transformation of strains with wcjE alleles from 11Av strains was sufficient to restore partial ßGal6OAc in an 11E background. We conclude that, instead of being distinct entities, serotypes 11A and 11E represent two extremes of an antigenic spectrum resulting from variable capsule O-acetylation secondary to heterologous wcjE mutations. These findings challenge whether all clinically relevant pneumococci can be definitively categorized into distinct serotypes.