Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2-Msh6 mismatch repair protein

Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1-but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.     

Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1–E1 and E1–E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.     

Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation

Glycoprotein B (gB) enables the fusion of viral and cell membranes during entry of herpesviruses. However, gB alone is insufficient for membrane fusion; the gH/gL heterodimer is also required. The crystal structure of the herpes simplex virus type 1 (HSV-1) gB ectodomain, gB730, has demonstrated similarities between gB and other viral fusion proteins, leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the membranes together by refolding from its initial or prefusion form to its final or postfusion form. The only available crystal structure likely represents the postfusion form of gB; the prefusion form has not yet been determined. Previously, a panel of HSV-1 gB mutants was generated by using random 5-amino-acid-linker insertion mutagenesis. Several mutants were unable to mediate cell-cell fusion despite being expressed on the cell surface. Mapping of the insertion sites onto the crystal structure of gB730 suggested that several insertions might not be accommodated in the postfusion form. Thus, we hypothesized that some insertion mutants were nonfunctional due to being “trapped” in a prefusion form. Here, we generated five insertion mutants as soluble ectodomains and characterized them biochemically. We show that the ectodomains of all five mutants assume conformations similar to that of the wild-type gB730. Four mutants have biochemical properties and overall structures that are indistinguishable from those of the wild-type gB730. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, one mutant, while able to adopt the overall postfusion structure, displays significant conformational differences in the vicinity of fusion loops, relative to wild-type gB730. Moreover, this mutant has a diminished ability to associate with liposomes, suggesting that the fusion loops in this mutant have decreased functional activity. We propose that these insertions cause a fusion-deficient phenotype not by preventing conversion of gB to a postfusion-like conformation but rather by interfering with other gB functions.Herpes simplex virus type 1 (HSV-1) is the prototype of the diverse herpesvirus family that includes many notable human pathogens (26). In addition to the icosahedral capsid and the tegument that surround its double-stranded DNA genome, herpesviruses have an envelope—an outer lipid bilayer—bearing a number of surface glycoproteins. During infection, HSV-1 must fuse its envelope with a cellular membrane in order to deliver the capsid into a target host cell. Among its viral glycoproteins, only glycoprotein C (gC), gB, gD, gH, and gL participate in this entry process, and only the last four are required for fusion (28). Although gD is found only in alphaherpesviruses, all herpesviruses encode gB, gH, and gL, which constitute their core fusion machinery. Of these three proteins, gB is the most highly conserved.We recently determined the crystal structure of a nearly full-length ectodomain of HSV-1 gB, gB730 (18). The crystal structure of the ectodomain of gB from Epstein-Barr virus, another herpesvirus, has also been subsequently determined (4). The two structures showed similarities between gB and other viral fusion proteins, in particular, G from an unrelated vesicular stomatitis virus (VSV) (25), leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the viral and host cell membranes together to enable their fusion. However, gB alone is known to be insufficient for membrane fusion; the gH/gL heterodimer is also required. This insufficiency raises the question of exactly how gB functions during viral entry. Answering this question is critical for understanding the complex mechanism that herpesviruses use to enter their host cells.In acting as a viral fusogen, gB must undergo dramatic conformational changes, refolding through a series of conformational intermediates from its initial, or prefusion form, to its final, or postfusion form (15). These conformational changes are not only necessary to bring the two membranes into proximity; they are also thought to provide the energy for the fusion process. The prefusion form corresponds to the protein present on the viral surface prior to initiation of fusion. The postfusion form represents the protein after fusion of the viral and host cell membranes. The available gB structure likely represents its postfusion form, since it shares more in common with the postfusion rather than the prefusion structure of vesicular stomatitis virus (VSV) G (3, 17). However, the prefusion form has not yet been characterized.Recently, a panel of gB mutants was generated by using random linker-insertion mutagenesis (21). Of these mutants, 16 were particularly interesting because they were nonfunctional in cell-cell fusion assays despite being expressed on the cell surface at levels that indicate proper folding for transport. These observations suggested that each insertion somehow interfered with gB function. Insertions in 12 of these mutants are located within the available structure of the gB ectodomain, which allowed Lin and Spear to analyze their locations (21).The most prominent examples of such nonfunctional mutants are two mutants with insertions after residues I185 or E187, henceforth referred to as “cavity mutants” because both I185 and E187 point into a cavity inside the gB trimer (Fig. 1B and D). Although this cavity might accommodate a single 5-amino-acid insertion, it “is not large enough to accommodate three 5-amino-acid insertions” (21) that would be present in the trimer (one insertion per protomer).Open in a separate windowFIG. 1.Location of the insertion sites in the sequence of gB and the structure of the postfusion form of its ectodomain. (A) Linear diagram of the full-length gB with functional domains highlighted (as in reference 18). Domain I is shown in cyan, domain II in green, domain III in yellow, domain IV in orange, domain V in red, and the disordered region between domains II and III in purple. Regions absent from the crystal structure of gB730 are shown in gray. Sequences in the region of 5-amino-acid insertions (residues 181 to 190 and residues 661 to 680) are shown in black. Arrows mark the locations of 5-amino-acid insertions, shown as red text. (B) Crystal structure of gB730 (18). Residues preceding the 5-amino-acid insertions in mutants studied here are shown as spheres colored by domain, consistent with panel A. Boxes delineate the hinge region, enlarged in panel C, and the cavity region, enlarged in panel D. (C) Close-up view of the hinge region shown in molecular surface representation, with residues 663 to 675 displayed as sticks. Hydrophobic residues are colored orange. Residues preceding the 5-amino-acid insertions in mutants studied here are labeled with asterisks; remaining labels correspond to additional hydrophobic residues in the 663-675 region. (D) Enlarged view of the cavity region. Residues that line the cavity and are not solvent exposed are colored magenta. Residue E187 of each protomer is colored teal and shown as spheres. Fusion loops for two protomers are marked with asterisks; the third pair of fusion loops lies behind the crystal structure and is not visible. Panels B, C, and D were made by using Pymol (http://www.pymol.org/).Five other nonfunctional mutants have insertions after residues D663, T665, V667, I671, or L673, respectively. We refer to them as “hinge mutants.” These residues lie in the region located between domains IV and V, which has been termed the hinge region because it may play an important role during the conformational transition from the prefusion to the postfusion form (17). Lin and Spear proposed that insertions following these residues “would likely affect hinge regions” (21), with the implication that they may prevent gB from refolding into the postfusion conformation. Our analysis suggested that insertions after these residues could, perhaps, be sterically accommodated in the structure but would probably be energetically unfavorable by causing several buried hydrophobic side chains in the 665-673 region, such as F670, I671, and L673, to become exposed (Fig. 1B and C).In light of these observations, we hypothesized that the insertion mutants are “trapped” in a prefusion form. We decided to test this hypothesis by determining whether the ectodomain of gB containing one of these insertion mutations is able to assume the conformation seen in the crystal structure of the wild-type gB ectodomain, which we are referring to as the likely postfusion conformation. For this purpose, we chose one cavity mutant, containing an insertion after E187, and four hinge mutants, containing insertions after T665, V667, I671, or L673, respectively. We chose to test four hinge mutants because structure analysis suggested to us that insertions following the respective residues might not affect the structure in precisely the same way. We expressed the soluble ectodomain of each mutant by using a baculovirus expression system and characterized the purified proteins by using biochemical and biophysical methods. Surprisingly, we found that the ectodomains of all five mutants assume a conformation similar to that of the wild-type gB ectodomain. The four hinge mutants had biochemical properties and overall three-dimensional structures that were indistinguishable from those of the wild-type gB ectodomain. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, the cavity mutant, while able to adopt the overall postfusion structure, still displayed significant conformational differences relative to wild-type gB. Because these conformational differences are in the vicinity of fusion loops, we conclude that the fusion loops in this mutant have decreased functional activity.     

Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX(4)D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.     

Cardiac fibrogenesis in magnesium deficiency: a role for circulating angiotensin II and aldosterone

Mechanisms underlying cardiac fibrogenesis in magnesium deficiency are unclear. It was reported earlier from this laboratory that serum from magnesium-deficient rats has a more pronounced stimulatory effect on cell proliferation, net collagen production, and superoxide generation in adult rat cardiac fibroblasts than serum from rats on the control diet. The profibrotic serum factors were, however, not identified. This study tested the hypothesis that circulating angiotensin II may modulate cardiac fibroblast activity in hypomagnesemic rats. Male Sprague-Dawley rats were pair-fed a magnesium-deficient (0.0008% Mg) or -sufficient (0.05%) diet for 6 days, and the effects of serum from these rats on [3H]thymidine and [3H]proline incorporation into cardiac fibroblasts from young adult rats were evaluated in the presence of losartan, an angiotensin II type 1 (AT1) receptor antagonist, and spironolactone, an aldosterone antagonist. Losartan and spironolactone markedly attenuated the stimulatory effects in vitro of serum from the magnesium-deficient and control groups, but the inhibitory effects were considerably higher in cells exposed to serum from magnesium-deficient animals. Circulating and cardiac tissue levels of angiotensin II were significantly elevated in magnesium-deficient animals (67.6% and 93.1%, respectively, vs. control). Plasma renin activity was 61.9% higher in magnesium-deficient rats, but serum angiotensin-converting enzyme activity was comparable in the two groups. Furthermore, preliminary experiments in vivo using enalapril supported a role for angiotensin II in magnesium deficiency. There was no significant difference between the groups in serum aldosterone levels. The findings suggest that circulating angiotensin II and aldosterone may stimulate fibroblast activity and contribute to a fibrogenic response in the heart in magnesium deficiency.     

Engineering proteins with enhanced mechanical stability by force-specific sequence motifs

Use of atomic force microscopy (AFM) has recently led to a better understanding of the molecular mechanisms of the unfolding process by mechanical forces; however, the rational design of novel proteins with specific mechanical strength remains challenging. We have approached this problem from a new perspective that generates linear physical–chemical properties (PCP) motifs from a limited AFM data set. Guided by our linear sequence analysis, we designed and analyzed four new mutants of the titin I1 domain with the goal of increasing the domain's mechanical strength. All four mutants could be cloned and expressed as soluble proteins. AFM data indicate that at least two of the mutants have increased molecular mechanical strength. This observation suggests that the PCP method is useful to graft sequences specific for high mechanical stability to weak proteins to increase their mechanical stability, and represents an additional tool in the design of novel proteins besides steered molecular dynamics calculations, coarse grained simulations, and ?‐value analysis of the transition state. Proteins 2012; © 2011 Wiley Periodicals, Inc.     

Identification of germline genomic copy number variation in familial pancreatic cancer

Adenocarcinoma of the pancreas is a significant cause of cancer mortality, and up to 10?% of cases appear to be familial. Heritable genomic copy number variants (CNVs) can modulate gene expression and predispose to disease. Here, we identify candidate predisposition genes for familial pancreatic cancer (FPC) by analyzing germline losses or gains present in one or more high-risk patients and absent in a large control group. A total of 120 FPC cases and 1,194 controls were genotyped on the Affymetrix 500K array, and 36 cases and 2,357 controls were genotyped on the Affymetrix 6.0 array. Detection of CNVs was performed by multiple computational algorithms and partially validated by quantitative PCR. We found no significant difference in the germline CNV profiles of cases and controls. A total of 93 non-redundant FPC-specific CNVs (53 losses and 40 gains) were identified in 50 cases, each CNV present in a single individual. FPC-specific CNVs overlapped the coding region of 88 RefSeq genes. Several of these genes have been reported to be differentially expressed and/or affected by copy number alterations in pancreatic adenocarcinoma. Further investigation in high-risk subjects may elucidate the role of one or more of these genes in genetic predisposition to pancreatic cancer.     

Base of the Measles Virus Fusion Trimer Head Receives the Signal That Triggers Membrane Fusion

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.