- Mitochondria as Molecular Platforms Integrating Multiple Innate Immune Signalings. [Review]
- JMJ Mol Biol 2016 Oct 27
- The immune system of vertebrates confers protective mechanisms to the host through the sensing of stress-induced agents expressed during infection or cell stress. Among them, the first line of host d...
The immune system of vertebrates confers protective mechanisms to the host through the sensing of stress-induced agents expressed during infection or cell stress. Among them, the first line of host defense composed of the innate immune sensing of these agents by pattern recognition receptors enables downstream adaptive immunity to be primed, mediating the body's appropriate response to clear infection and tissue damage. Mitochondria are «bacteria within» that allowed the emergence of functional eukaryotic cells by positioning themselves as the cell powerhouse and an initiator of cell death programs. It is striking to consider that such ancestral bacteria, which had to evade host defense at some point to develop evolutionary endosymbiosis, have become instrumental for the modern eukaryotic cell in alerting the immune system against various insults including infection by other pathogens. Mitochondria have indeed become critical regulators of innate immune responses to both pathogens and cell stress. They host numerous modulators, which play a direct role into the assembly of innate sensing machineries that trigger host immune response in both sterile and non-sterile conditions. Several lines of evidence indicate the existence of a complex molecular interplay between mechanisms involved in inflammation and metabolism. Mitochondrial function seems to participate in innate immunity at various stages as diverse as the transcriptional regulation of inflammatory cytokines and chemokines and their maturation by inflammasomes. Here, we review the mechanisms by which mitochondria orchestrate innate immune responses at different levels by promoting a cellular metabolic reprogramming and the cytosolic immune signaling cascades.
- Deciphering, communicating, and engineering the CRISPR PAM. [Review]
- JMJ Mol Biol 2016 Dec 01
- Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their flanking CRISPR-associated (cas) genes make up RNA-guided, adaptive immune systems in prokaryotes whose effector prote...
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their flanking CRISPR-associated (cas) genes make up RNA-guided, adaptive immune systems in prokaryotes whose effector proteins have become powerful tools for basic research and biotechnology. While the Cas effector proteins are remarkably diverse, they commonly rely on protospacer-adjacent motifs (PAMs) as the first step in target recognition. PAM sequences are known to vary considerably between systems and have proven to be difficult to predict, spurring the need for new tools to rapidly identify and communicate these sequences. Recent advances have also shown that Cas proteins can be engineered to alter PAM recognition, opening new opportunities to develop CRISPR-based tools with enhanced targeting capabilities. In this review, we discuss the properties of the CRISPR PAM and the emerging tools for determining, visualizing, and engineering PAM recognition. We also propose a standard means of orienting the PAM to simplify how its location and sequence are communicated.
- PIC activation through functional interplay between Mediator and TFIIH. [Journal Article]
- JMJ Mol Biol 2016 Dec 01
- The multiprotein Mediator coactivator complex functions in large part by controlling formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II (Pol...
The multiprotein Mediator coactivator complex functions in large part by controlling formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II (Pol II) and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process.
- SCOPe: Manual Curation and Artifact Removal in the Structural Classification of Proteins - extended Database. [Journal Article]
- JMJ Mol Biol 2016 Nov 30
- SCOPe (Structural Classification of Proteins - extended, http://scop.berkeley.edu) is a database of relationships between protein structures that extends the Structural Classification of Proteins (SC...
SCOPe (Structural Classification of Proteins - extended, http://scop.berkeley.edu) is a database of relationships between protein structures that extends the Structural Classification of Proteins (SCOP) database. SCOP is an expert-curated ordering of domains from the majority of proteins of known structure in a hierarchy according to structural and evolutionary relationships. SCOPe classifies the majority of protein structures released since SCOP development concluded in 2009, using a combination of manual curation and highly precise automated tools, aiming to have the same accuracy as fully hand-curated SCOP releases. SCOPe also incorporates and updates the ASTRAL compendium, which provides several databases and tools to aid in the analysis of the sequences and structures of proteins classified in SCOPe. SCOPe continues high quality manual classification of new superfamilies, a key feature of SCOP. Artifacts such as expression tags are now separated into their own class, in order to distinguish them from the homology-based annotations in the remainder of the SCOPe hierarchy. SCOPe 2.06 contains 77,439 PDB entries, double the 38,221 structures classified in SCOP.
- Near-atomic resolution structure determination of a cypovirus capsid and polymerase complex using cryo-EM at 200kV. [Journal Article]
- JMJ Mol Biol 2016 Nov 30
- Single particle cryo-electron microscopy (cryo-EM) allows high-resolution structural determination of biological assemblies in a near-native environment. However, all high-resolution (better than 3.5...
Single particle cryo-electron microscopy (cryo-EM) allows high-resolution structural determination of biological assemblies in a near-native environment. However, all high-resolution (better than 3.5Å) cryo-EM structures reported to date were obtained by using 300kV transmission electron microscopes (TEMs). We report here the structures of a cypovirus capsid of 750Å diameter at 3.3Å resolution and RNA-dependent RNA polymerase (RdRp) complexes within the capsid at 3.9Å resolution using a 200kV TEM. The newly resolved structure revealed conformational changes of two subdomains in the RdRp. These conformational changes, which were involved in RdRp's switch from non-transcribing to transcribing mode, suggest that the RdRp may facilitate the unwinding of genomic double-stranded RNA. The possibility of 3Å resolution structural determinations for biological assemblies of relatively small sizes using cryo-EM at 200kV was discussed.
- Multiple Conformations of Gal3 Protein Drive the Galactose Induced Allosteric Activation of the GAL Genetic Switch of Saccharomyces cerevisiae. [Journal Article]
- JMJ Mol Biol 2016 Nov 29
- Gal3p is an allosteric monomeric protein which activates the GAL genetic switch of Saccharomyces cerevisiae in response to galactose. Expression of constitutive mutant of Gal3p or overexpression of w...
Gal3p is an allosteric monomeric protein which activates the GAL genetic switch of Saccharomyces cerevisiae in response to galactose. Expression of constitutive mutant of Gal3p or overexpression of wild-type Gal3p activates the GAL switch in the absence of galactose. These data suggest that Gal3p exists as an ensemble of active and inactive conformations. Structural data has indicated that Gal3p exists in open (inactive) and closed (active) conformations. However, mutant of Gal3p that predominantly exists in inactive conformation and yet capable of responding to galactose has not been isolated. To understand the mechanism of allosteric transition, we have isolated a triple mutant of Gal3p with V273I, T404A and N450D substitutions which upon overexpression fails to activate the GAL switch on its own, but activates the switch in response to galactose. Overexpression of Gal3p mutants with single or double mutations in any of the three combinations failed to exhibit the behavior of the triple mutant. Molecular dynamics analysis of the wild-type and the triple mutant along with two previously reported constitutive mutants suggest that the wild-type Gal3p may also exist in super-open conformation. Further, our results suggest that the dynamics of residue F237 situated in the hydrophobic pocket located in the hinge region drives the transition between different conformations. Based on this study, we suggest that conformational selection mechanism is the driving force in the allosteric transition of Gal3p, which may have implications in other signaling pathways involving monomeric proteins.
- Free Energy Perturbation Calculation of Relative Binding Free energy between Broadly Neutralizing Antibodies and the gp120 Glycoprotein of HIV-1. [Journal Article]
- JMJ Mol Biol 2016 Nov 28
- Direct calculation of relative binding affinities between antibodies and antigens is a long-sought goal. However, despite substantial efforts, no generally applicable computational method has been de...
Direct calculation of relative binding affinities between antibodies and antigens is a long-sought goal. However, despite substantial efforts, no generally applicable computational method has been described. Here we describe a systematic free energy perturbation (FEP) protocol and calculate the binding affinities between the gp120 envelope glycoprotein of HIV-1 and three broadly neutralizing antibodies (bNAbs) of the VRC01 class. The protocol has been adapted from successful studies of small molecules to address the challenges associated with modeling protein-protein interactions. Specifically, we built homology models of the three antibody-gp120 complexes, extended sampling times for large bulky residues, incorporated modeling of glycans on the surface of gp120, and utilized continuum solvent-based loop prediction protocols to improve sampling. We present three experimental surface plasmon resonance data sets, in which antibody residues in the antibody/gp120 interface were systematically mutated to alanine. The RMS error in the large set (55 total cases) of FEP tests as compared to these experiments, 0.68kcal/mol, is near experimental accuracy, and compares favorably with results obtained from a simpler, empirical methodology. The correlation coefficient for the combined data set including residues with glycan contacts, R(2)=0.48, should be sufficient to guide the choice of residues for antibody optimization projects, assuming this level of accuracy can be realized in prospective prediction. More generally, these results are encouraging with regard to the possibility of using an FEP approach to calculate the magnitude of protein-protein binding affinities.
- BindProfX: Assessing Mutation-Induced Binding Affinity Change by Protein Interface Profiles with Pseudo-Counts. [Journal Article]
- JMJ Mol Biol 2016 Nov 27
- Understanding how gene-level mutations affect the binding affinity of protein-protein interactions is a key issue of protein engineering. Due to the complexity of the problem, using physical force fi...
Understanding how gene-level mutations affect the binding affinity of protein-protein interactions is a key issue of protein engineering. Due to the complexity of the problem, using physical force field to predict the mutation-induced binding free-energy change remains challenging. In this work, we present a renewed approach to calculate the impact of gene mutations on the binding affinity through the structure-based profiling of protein-protein interfaces, where the binding free-energy change (ΔΔG) is counted as the logarithm of relative probability of mutant amino acids over wild-type ones in the interface alignment matrix; three pseudo-counts are introduced to alleviate the limit of the current interface library. Compared with a previous profile score that was based on the log-odds likelihood calculation, the correlation between predicted and experimental ΔΔG of single-site mutations is increased in this approach from 0.33 to 0.68. The structure-based profile score is found complementary to the physical potentials, where a linear combination of the profile score with the FoldX potential could increase the ΔΔG correlation from 0.46 to 0.74. It is also shown that the profile score is robust for counting the coupling effect of multiple individual mutations. For the mutations involving more than two mutation sites where the correlation between FoldX and experimental data vanishes, the profile-based calculation retains a strong correlation with the experimental measurements.
- The histone variant H3.3 in transcriptional regulation and human disease. [Review]
- JMJ Mol Biol 2016 Nov 25
- Histone proteins wrap around DNA to form nucleosomes, which further compact into higher order structure of chromatin. In addition to the canonical histones, there are also variant histones that often...
Histone proteins wrap around DNA to form nucleosomes, which further compact into higher order structure of chromatin. In addition to the canonical histones, there are also variant histones that often have pivotal roles in regulating chromatin dynamics and the accessibility of the underlying DNA. H3.3 is the most common non-centromeric variant of histone H3 that differs from the canonical H3 by just 4-5 amino acids. Here we discuss the current knowledge of H3.3 in transcriptional regulation and the recent discoveries and molecular mechanisms of H3.3 mutations in human cancer.
New Search Next
- Identifying residues that determine SCF molecular-level interactions through a combination of experimental and in silico analyses. [Journal Article]
- JMJ Mol Biol 2016 Nov 24
- The stem cell factor (SCF)/c-Kit receptor tyrosine kinase complex - with its significant roles in hematopoiesis and angiogenesis - is an attractive target for rational drug design. There is thus a ne...
The stem cell factor (SCF)/c-Kit receptor tyrosine kinase complex - with its significant roles in hematopoiesis and angiogenesis - is an attractive target for rational drug design. There is thus a need to map, in detail, the SCF/c-Kit interaction sites and the mechanisms that modulate this interaction. While most residues in the direct SCF/c-Kit binding interface can be identified from the existing crystal structure of the complex, other residues that affect binding through protein unfolding, intermolecular interactions, or allosteric or long-distance electrostatic effects cannot be directly inferred. Here, we describe an efficient method for protein-wide epitope mapping using yeast surface display (YSD). A library of single SCF mutants that span the SCF sequence was screened for decreased affinity to soluble c-Kit. Sequencing of selected clones allowed identification of mutations that reduce SCF binding affinity to c-Kit. Moreover, screening of these SCF clones for binding to a structural antibody, helped to identify mutations that result in small or large conformational changes in SCF. Computational modeling of the experimentally identified mutations showed that these mutations reduced the binding affinity through one of three scenarios: through SCF destabilization, through elimination of favorable SCF/c-Kit intermolecular interactions, or through allosteric changes. Eight SCF variants were expressed and purified. Experimentally measured in vitro binding affinities of these mutants to c-Kit confirmed both the YSD selection results and the computational predictions. This study has thus identified the residues crucial for c-Kit/SCF binding and has demonstrated the advantages of using a combination of computational and combinatorial methods for epitope mapping.