Satyan Sharma

Proteome-wide Analysis of Lysine Acetylation Suggests its Broad Regulatory Scope in Saccharomyces cerevisiae

Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with nonacetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites identified several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes.

Structural Enzymological Studies of 2-Enoyl Thioester Reductase of the Human Mitochondrial Fas II Pathway: New Insights Into its Substrate Recognition Properties.

Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway.

Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging.

Theoretical investigations of prostatic acid phosphatase

The phosphotyrosyl protein phosphatase activity of prostatic acid phosphatase (PAP) has been well established. It has also been suggested that PAP partly regulates the activity of growth factor receptors by dephosphorylating the autophosphorylysable tyrosines in them. We studied the binding of the peptides from epidermal growth factor receptor (EGFR) and its homolog (ErbB‐2), corresponding to their autophosphorylation sites, to PAP using theoretical modeling and molecular dynamics (MD) simulation methods. Nine different peptides, each with a phosphotyrosine residue, were docked on human PAP. The binding energies of these peptide–PAP complexes were calculated theoretically and compared to experimentally obtained affinities. The peptide AceDNLpYYWDNH2 from ErbB‐2(1197–1203) showed the most favorable free energy of binding when estimated theoretically. The results demonstrate that the presence of another tyrosine residue proximate to C‐terminal of autophosphorylysable Tyr enhances the binding affinity considerably. The presence of a bulky group instead prevents the binding, as is observed in case of peptide AceNLYpYWDQNH2 which failed to bind, both in theoretical calculations and experiments. Thus we demonstarted that PAP could potentially bind to EGFR and Erbb‐2 and dephosphorylate them. Thus it could be involved in the regulation of the function of such receptors. In addition, complexes of a peptide from AngiotensinII and phosphotyrosine(pY) with human PAP were also modeled. The effects of different protonation states of the titratable active site residues on ligand (pY) binding have also been investigated. For a favorable binding His12 and Asp258 should be neutral, His257 should be positively charged and the phosphate group of the ligand should be in PO  43− state. Furthermore, the analysis of protein motion as observed during simulations suggests the loop–loop contact in the PAP dimer to be of importance in cooperativity. Proteins 2005.

A novel mutation of the fumarase gene in a family with autosomal recessive fumarase deficiency

Fumarase hydratase (FH) deficiency is a rare familial disorder of the tricarboxylic acid cycle which is characterized by severe neurological impairment in early childhood. Several autosomal recessive mutations in the fumarate hydratase gene have been identified as a cause of the lack of fumarase activity in affected individuals. We describe a novel mutation in nucleotide 1127A>C of the fumarase cDNA which changes glutamine 376 to proline in the vicinity of the catalytic site and explains the loss of FH function. Two homozygous carriers of this mutation suffered from severe encephalopthy and died at a young age. Molecular modeling of FH structure shows that the mutation Gln376Pro in the second half of the fumarase sequence disrupts the structure of the active site. Analysis of the FH mutation and the mutant enzyme variant described here provides an explanation for the mechanism of FH deficiency at the molecular level and paves the way for the analysis of other dysfunctional FH variants.

v-SNARE transmembrane domains function as catalysts for vesicle fusion

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion. DOI: http://dx.doi.org/10.7554/eLife.17571.001

A DFT Study on the Formation of a Phosphohistidine Intermediate in Prostatic Acid Phosphatase

Histidine phosphatases are a class of enzymes that are characterized by the presence of a conserved RHGXRXP motif. This motif contains a catalytic histidine that is being phosphorylated in the course of a dephosphorylation reaction catalyzed by these enzymes. Prostatic acid phosphatase (PAP) is one such enzyme. The dephosphorylation of phosphotyrosine by PAP is a two-step process. The first step involves the transfer of a phosphate group from the substrate to the histidine (His12). The present study reports on the details of the first step of this reaction, which was investigated using a series of quantum chemistry calculations. A number of quantum models were constructed containing various residues that were thought to play a role in the mechanism. In all these models, the transition state displayed an associative character. The transition state is stabilized by three active site arginines (Arg11, Arg15, and Arg79), two of which belong to the aforementioned conserved motif. The work also demonstrated that His12 could act as a nucleophile. The enzyme is further characterized by a His257-Asp258 motif. The role of Asp258 has been elusive. In this work, we propose that Asp258 acts as a proton donor which becomes protonated when the substrate enters the binding pocket. Evidence is also obtained that the transfer of a proton from Asp258 to the leaving group is possibly mediated by a water molecule in the active site. The work also underlines the importance of His257 in lowering the energy barrier for the nucleophilic attack.

A coarse grained model for a lipid membrane with physiological composition and leaflet asymmetry.

The resemblance of lipid membrane models to physiological membranes determines how well molecular dynamics (MD) simulations imitate the dynamic behavior of cell membranes and membrane proteins. Physiological lipid membranes are composed of multiple types of phospholipids, and the leaflet compositions are generally asymmetric. Here we describe an approach for self-assembly of a Coarse-Grained (CG) membrane model with physiological composition and leaflet asymmetry using the MARTINI force field. An initial set-up of two boxes with different types of lipids according to the leaflet asymmetry of mammalian cell membranes stacked with 0.5 nm overlap, reliably resulted in the self-assembly of bilayer membranes with leaflet asymmetry resembling that of physiological mammalian cell membranes. Self-assembly in the presence of a fragment of the plasma membrane protein syntaxin 1A led to spontaneous specific positioning of phosphatidylionositol(4,5)bisphosphate at a positively charged stretch of syntaxin consistent with experimental data. An analogous approach choosing an initial set-up with two concentric shells filled with different lipid types results in successful assembly of a spherical vesicle with asymmetric leaflet composition. Self-assembly of the vesicle in the presence of the synaptic vesicle protein synaptobrevin 2 revealed the correct position of the synaptobrevin transmembrane domain. This is the first CG MD method to form a membrane with physiological lipid composition as well as leaflet asymmetry by self-assembly and will enable unbiased studies of the incorporation and dynamics of membrane proteins in more realistic CG membrane models.

An atomistic model for assembly of transmembrane domain of T cell receptor complex.

The T cell receptor (TCR) together with accessory cluster of differentiation 3 (CD3) molecules (TCR-CD3 complex) is a key component in the primary function of T cells. The nature of association of the transmembrane domains is of central importance to the assembly of the complex and is largely unknown. Using multiscale molecular modeling and simulations, we have investigated the structure and assembly of the TCRα-CD3ε-CD3δ transmembrane domains both in membrane and in micelle environments. We demonstrate that in a membrane environment the transmembrane basic residue of the TCR closely interacts with both of the transmembrane acidic residues of the CD3 dimer. In contrast, in a micelle the basic residue interacts with only one of the acidic residues. Simulations of a recent micellar nuclear magnetic resonance structure of the natural killer (NK) cell-activating NKG2C-DAP12-DAP12 trimer in a membrane further indicate that the environment significantly affects the way these trimers associate. Since the currently accepted model for transmembrane association is entirely based on a micellar structure, we propose a revised model for the association of transmembrane domains of the activating immune receptors in a membrane environment.

The mystery of the fusion pore

Release of neurotransmitters occurs by opening of a fusion pore in a manner thought to be mediated by SNARE proteins, but whether the fusion pore is a lipidic or a proteinaceous structure is controversial. A new study using very small nanodiscs shows that it is both.