Publications by authors named "Christopher E Berndsen"

32 Publications

Solution structure and assembly of β-amylase 2 from Arabidopsis thaliana.

Acta Crystallogr D Struct Biol 2020 Apr 25;76(Pt 4):357-365. Epub 2020 Mar 25.

Department of Chemistry and Biochemistry, James Madison University, 901 Carrier Drive, MSC 4501, Harrisonburg, VA 22807, USA.

Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. β-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.
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http://dx.doi.org/10.1107/S2059798320002016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137110PMC
April 2020

Resilience of BST-2/Tetherin structure to single amino acid substitutions.

PeerJ 2019 31;7:e7043. Epub 2019 May 31.

Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA, United States of America.

Human tetherin, also known as BST-2 or CD317, is a dimeric, extracellular membrane-bound protein that consists of N and C terminal membrane anchors connected by an extracellular domain. BST-2 is involved in binding enveloped viruses, such as HIV, and inhibiting viral release in addition to a role in NF-kB signaling. Viral tethering by tetherin can be disrupted by the interaction with Vpu in HIV-1 in addition to other viral proteins. The structural mechanism of tetherin function is not clear and the effects of human tetherin mutations identified by sequencing consortiums are not known. To address this gap in the knowledge, we used data from the Ensembl database to construct and model known human missense mutations within the ectodomain to investigate how the structure of the ectodomain influences function. From the data, we identified an island of sequence stability within the ectodomain, which corresponds to a functionally and structurally important region identified in previous biochemical and biophysical studies. Most of the modeled mutations had little effect on the structure of the dimer and the coiled-coil, suggesting that the coiled-coil compensates for changes in primary structure. Thus, many of the functional defects observed in previous studies may not be due to changes in tetherin structure, but rather, due to in changes in protein-protein interactions or in aspects of tetherin not currently understood. The lack of structural effects by mutations known to decrease function further illustrates the need for more study of the structure-function connection for this system. Finally, apparent flexibility in tetherin sequence may allow for greater anti-viral activities with a larger number of viruses by reducing specific interactions with anti-tetherin proteins, while maintaining virus restriction.
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http://dx.doi.org/10.7717/peerj.7043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546079PMC
May 2019

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of β-AMYLASE2 From .

Front Plant Sci 2018 14;9:1176. Epub 2018 Aug 14.

Department of Biology, Western Carolina University, Cullowhee, NC, United States.

The β-amylase family in has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a similar to that of the wild type enzyme but with a 7.5-fold lower for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.
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http://dx.doi.org/10.3389/fpls.2018.01176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102588PMC
August 2018

In silico modeling of epigenetic-induced changes in photoreceptor cis-regulatory elements.

Mol Vis 2018 14;24:218-230. Epub 2018 Mar 14.

Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA.

Purpose: DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements.

Methods: We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of , , and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human regulatory sequences.

Results: In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human and genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within regulatory sequences which may account for the repression of CRX-dependent transcription.

Conclusions: Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851326PMC
November 2018

Glutathionylation Inhibits the Catalytic Activity of Arabidopsis β-Amylase3 but Not That of Paralog β-Amylase1.

Biochemistry 2018 02 22;57(5):711-721. Epub 2018 Jan 22.

Department of Biology, James Madison University , Harrisonburg, Virginia 22807, United States.

β-Amylase3 (BAM3) is an enzyme that is essential for starch degradation in plant leaves and is also transcriptionally induced under cold stress. However, we recently reported that BAM3's enzymatic activity decreased in cold-stressed Arabidopsis leaves, although the activity of BAM1, a homologous leaf β-amylase, was largely unaffected. This decrease in BAM3 activity may relate to the accumulation of starch reported in cold-stressed plants. The aim of this study was to explore the disparity between BAM3 transcript and activity levels under cold stress, and we present evidence suggesting BAM3 is being inhibited by post-translational modification. A mechanism of enzyme inhibition was suggested by observing that BAM3 protein levels remained unchanged under cold stress. Cold stress induces nitric oxide (NO) signaling, one result being alteration of protein activity by nitrosylation or glutathionylation through agents such as S-nitrosoglutathione (GSNO). To test whether NO induction correlates with inhibition of BAM3 in vivo, plants were treated with sodium nitroprusside, which releases NO, and a decline in BAM3 but not BAM1 activity was again observed. Treatment of recombinant BAM3 and BAM1 with GSNO caused significant, dose-dependent inhibition of BAM3 activity while BAM1 was largely unaffected. Site-directed mutagenesis, anti-glutathione Western blots, and mass spectrometry were then used to determine that in vitro BAM3 inhibition was caused by glutathionylation at cysteine 433. In addition, we generated a BAM1 mutant resembling BAM3 that was sensitive to GSNO inhibition. These findings demonstrate a differential response of two BAM paralogs to the Cys-modifying reagent GSNO and provide a possible molecular basis for reduced BAM3 activity in cold-stressed plants.
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http://dx.doi.org/10.1021/acs.biochem.7b01274DOI Listing
February 2018

A novel FLNC frameshift and an OBSCN variant in a family with distal muscular dystrophy.

PLoS One 2017 26;12(10):e0186642. Epub 2017 Oct 26.

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena and Azienda Ospedaliera Universitaria Senese, Siena, Italy.

A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186642PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657976PMC
November 2017

Arabidopsis β-Amylase2 Is a K-Requiring, Catalytic Tetramer with Sigmoidal Kinetics.

Plant Physiol 2017 Dec 24;175(4):1525-1535. Epub 2017 Oct 24.

Department of Biology, James Madison University, Harrisonburg, Virginia 22807.

The Arabidopsis () genome contains nine β-amylase () genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.
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http://dx.doi.org/10.1104/pp.17.01506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717748PMC
December 2017

Novel obscurins mediate cardiomyocyte adhesion and size via the PI3K/AKT/mTOR signaling pathway.

J Mol Cell Cardiol 2017 10 4;111:27-39. Epub 2017 Aug 4.

Department of Biochemistry and Molecular Biology, University of Maryland, School of Medicine, Baltimore, MD 21201, United States. Electronic address:

The intercalated disc of cardiac muscle embodies a highly-ordered, multifunctional network, essential for the synchronous contraction of the heart. Over 200 known proteins localize to the intercalated disc. The challenge now lies in their characterization as it relates to the coupling of neighboring cells and whole heart function. Using molecular, biochemical and imaging techniques, we characterized for the first time two small obscurin isoforms, obscurin-40 and obscurin-80, which are enriched at distinct locations of the intercalated disc. Both proteins bind specifically and directly to select phospholipids via their pleckstrin homology (PH) domain. Overexpression of either isoform or the PH-domain in cardiomyocytes results in decreased cell adhesion and size via reduced activation of the PI3K/AKT/mTOR pathway that is intimately linked to cardiac hypertrophy. In addition, obscurin-80 and obscurin-40 are significantly reduced in acute (myocardial infarction) and chronic (pressure overload) murine cardiac-stress models underscoring their key role in maintaining cardiac homeostasis. Our novel findings implicate small obscurins in the maintenance of cardiomyocyte size and coupling, and the development of heart failure by antagonizing the PI3K/AKT/mTOR pathway.
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http://dx.doi.org/10.1016/j.yjmcc.2017.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5694667PMC
October 2017

Bending of the BST-2 coiled-coil during viral budding.

Proteins 2017 Nov 12;85(11):2081-2087. Epub 2017 Aug 12.

Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia.

BST-2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV-1 and other viruses by inhibiting viral spreading. Structurally, BST-2 is a homo-dimeric coiled-coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C-terminal membrane anchor of BST-2 is inserted into the budding virus while the N-terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST-2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST-2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated form to the cell-virus membrane bridging form. We observed that BST-2 did not transition as a rigid structure, but instead bent at positions with a reduced interface between the helices of the coiled-coil. The simulations for the human BST-2 were then compared with simulations on the mouse homolog, which has no apparent weak spots. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled-coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.
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http://dx.doi.org/10.1002/prot.25362DOI Listing
November 2017

Deregulated Ca cycling underlies the development of arrhythmia and heart disease due to mutant obscurin.

Sci Adv 2017 06 7;3(6):e1603081. Epub 2017 Jun 7.

Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Baltimore, MD 21201, USA.

Obscurins are cytoskeletal proteins with structural and regulatory roles encoded by . Mutations in are associated with the development of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Specifically, the R4344Q mutation present in immunoglobulin domain 58 (Ig58) was the first to be linked with the development of HCM. To assess the effects of R4344Q in vivo, we generated the respective knock-in mouse model. Mutant obscurins are expressed and incorporated normally into sarcomeres. The expression patterns of sarcomeric and Ca-cycling proteins are unaltered in sedentary 1-year-old knock-in myocardia, with the exception of sarco/endoplasmic reticulum Ca adenosine triphosphatase 2 (SERCA2) and pentameric phospholamban whose levels are significantly increased and decreased, respectively. Isolated cardiomyocytes from 1-year-old knock-in hearts exhibit increased Ca-transients and Ca-load in the sarcoplasmic reticulum and faster contractility kinetics. Moreover, sedentary 1-year-old knock-in animals develop tachycardia accompanied by premature ventricular contractions, whereas 2-month-old knock-in animals subjected to pressure overload develop a DCM-like phenotype. Structural analysis revealed that the R4344Q mutation alters the distribution of electrostatic charges over the Ig58 surface, thus interfering with its binding capabilities. Consistent with this, wild-type Ig58 interacts with phospholamban modestly, and this interaction is markedly enhanced in the presence of R4344Q. Together, our studies demonstrate that under sedentary conditions, the R4344Q mutation results in Ca deregulation and spontaneous arrhythmia, whereas in the presence of chronic, pathological stress, it leads to cardiac remodeling and dilation. We postulate that enhanced binding between mutant obscurins and phospholamban leads to SERCA2 disinhibition, which may underlie the observed pathological alterations.
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http://dx.doi.org/10.1126/sciadv.1603081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462502PMC
June 2017

Novel insights into the interaction of UBA5 with UFM1 via a UFM1-interacting sequence.

Sci Rep 2017 03 30;7(1):508. Epub 2017 Mar 30.

Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.

The modification of proteins by ubiquitin-fold modifier 1 (UFM1) is implicated in many human diseases. Prior to conjugation, UFM1 undergoes activation by its cognate activating enzyme, UBA5. UBA5 is a non-canonical E1 activating enzyme that possesses an adenylation domain but lacks a distinct cysteine domain. Binding of UBA5 to UFM1 is mediated via an amino acid sequence, known as the UFM1-interacting sequence (UIS), located outside the adenylation domain that is required for UFM1 activation. However, the precise boundaries of the UIS are yet not clear and are still under debate. Here we revisit the interaction of UFM1 with UBA5 by determining the crystal structure of UFM1 fused to 13 amino acids of human UBA5. Using binding and activity assays, we found that His 336 of UBA5, previously not reported to be part of the UIS, occupies a negatively charged pocket on UFM1's surface. This His is involved in UFM1 binding and if mutated perturbs activation of UFM1. Surprisingly, we also found that the interaction between two UFM1 molecules mimics how the UIS binds UFM1. Specifically, UFM1 His 70 resembles UBA5 His336 and enters a negatively charged pocked on the other UFM1 molecule. Our results refine our understanding of UFM1-UBA5 binding.
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http://dx.doi.org/10.1038/s41598-017-00610-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428781PMC
March 2017

Trans-Binding Mechanism of Ubiquitin-like Protein Activation Revealed by a UBA5-UFM1 Complex.

Cell Rep 2016 09;16(12):3113-3120

Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. Electronic address:

Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s). Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans-binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation.
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http://dx.doi.org/10.1016/j.celrep.2016.08.067DOI Listing
September 2016

Characterization of the structure and catalytic activity of Legionella pneumophila VipF.

Proteins 2016 10 5;84(10):1422-30. Epub 2016 Jul 5.

Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia, 22807.

The pathogenic bacteria Legionella pneumophila is known to cause Legionnaires' Disease, a severe pneumonia that can be fatal to immunocompromised individuals and the elderly. Shohdy et al. identified the L. pneumophila vacuole sorting inhibitory protein VipF as a putative N-acetyltransferase based on sequence homology. We have characterized the basic structural and functional properties of VipF to confirm this original functional assignment. Sequence conservation analysis indicates two putative CoA-binding regions within VipF. Homology modeling and small angle X-ray scattering suggest a monomeric, dual-domain structure joined by a flexible linker. Each domain contains the characteristic beta-splay motif found in many acetyltransferases, suggesting that VipF may contain two active sites. Docking experiments suggest reasonable acetyl-CoA binding locations within each beta-splay motif. Broad substrate screening indicated that VipF is capable of acetylating chloramphenicol and both domains are catalytically active. Given that chloramphenicol is not known to be N-acetylated, this is a surprising finding suggesting that VipF is capable of O-acetyltransferase activity. Proteins 2016; 84:1422-1430. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25087DOI Listing
October 2016

Connecting common genetic polymorphisms to protein function: A modular project sequence for lecture or lab.

Biochem Mol Biol Educ 2016 Nov 9;44(6):526-536. Epub 2016 Jun 9.

Center for Genome and Metagenome Studies, James Madison University, Virginia.

Single nucleotide polymorphisms (SNPs) in DNA can result in phenotypes where the biochemical basis may not be clear due to the lack of protein structures. With the growing number of modeling and simulation software available on the internet, students can now participate in determining how small changes in genetic information impact cellular protein structure and function. We have developed a modular series of activities to engage lab or lecture students in examining the basis for common phenotypes. The activities range from basic phenotype testing/observation to DNA sequencing and simulation of protein structure and dynamics. We provide as an example study of the bitterness receptor TAS2R38 and PTC tasting, however these activities are applicable to other SNPs or genomic variants with a direct connection to an observable phenotype. These activities are modular and can be mixed to meet the student capabilities and infrastructure availability. The complete sequence of activities will demonstrate the direct connection between gene structure, protein structure and organism function. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):526-536, 2016.
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http://dx.doi.org/10.1002/bmb.20976DOI Listing
November 2016

The Disulfide Bonds within BST-2 Enhance Tensile Strength during Viral Tethering.

Biochemistry 2016 Feb 4;55(6):940-7. Epub 2016 Feb 4.

Department of Chemistry and Biochemistry, James Madison University , Harrisonburg, Virginia 22807, United States.

Human BST-2/tetherin is a host factor that inhibits the release of enveloped viruses, including HIV-1, HIV-2, and SIV, from the cell surface by tethering viruses to the host cell membrane. BST-2 has an α-helical ectodomain that forms disulfide-linked dimers between two monomers forming a coiled coil. The ectodomain contains three cysteine residues that can participate in disulfide bond formation and are critical for viral tethering. The role of the disulfides in viral tethering is unknown but proposed to be for maintaining the dimer. We explored the role of the disulfides in the structure of BST-2 using experimental, biophysical methods. To understand the role of the disulfides in viral tethering, we used a new approach in viral tethering, steered molecular dynamics. We find that the disulfides coordinate the unfolding of the BST-2 monomers, which adds tensile strength to the coiled coil. Structural differences between oxidized and reduced BST-2 are apparent during unfolding, showing the monomers slide past each other in the absence of the disulfides. We found no evidence to support dissociation of the dimer upon reduction of the disulfide bonds. Moreover, the structure of BST-2 in the absence of the disulfides is similar to that of the oxidized form of BST-2, supporting previous X-ray crystallography and cellular work that showed the disulfides are not required for expression of BST-2. These data provide new insights into viral tethering by using novel techniques in the analysis of BST-2 to give amino acid level insight into functions of BST-2.
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http://dx.doi.org/10.1021/acs.biochem.5b01362DOI Listing
February 2016

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage.

J Biol Chem 2016 Jan 24;291(4):2033-2042. Epub 2015 Nov 24.

From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and. Electronic address:

The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys(63)-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys(63)-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys(63)-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage.
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http://dx.doi.org/10.1074/jbc.M115.689869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722476PMC
January 2016

Chemical shift assignments for S. cerevisiae Ubc13.

Biomol NMR Assign 2015 Oct 7;9(2):407-10. Epub 2015 May 7.

Department of Chemistry and Biochemistry, James Madison University, 901 Carrier Dr., Harrisonburg, VA, 22807, USA.

The ubiquitination pathway controls several human cellular processes, most notably protein degradation. Ubiquitin, a small signaling protein, is activated by the E1 activating enzyme, transferred to an E2 conjugating enzyme, and then attached to a target substrate through a process that can be facilitated by an E3 ligase enzyme. The enzymatic mechanism of ubiquitin transfer from the E2 conjugating enzyme onto substrate is not clear. The highly conserved HPN motif in E2 catalytic domains is generally thought to help stabilize an oxyanion intermediate formed during ubiquitin transfer. However recent work suggests this motif is instead involved in a structural, non-enzymatic role. As a platform to better understand the E2 catalyzed ubiquitin transfer mechanism, we determined the chemical shift assignments of S. cerevisiae E2 enzyme Ubc13.
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http://dx.doi.org/10.1007/s12104-015-9619-xDOI Listing
October 2015

Positioning of cysteine residues within the N-terminal portion of the BST-2/tetherin ectodomain is important for functional dimerization of BST-2.

J Biol Chem 2015 Feb 18;290(6):3740-51. Epub 2014 Dec 18.

From the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460 and

BST-2/tetherin is a cellular host factor capable of restricting the release of a variety of enveloped viruses, including HIV-1. Structurally, BST-2 consists of an N-terminal cytoplasmic domain, a transmembrane domain, an ectodomain, and a C-terminal membrane anchor. The BST-2 ectodomain encodes three cysteine residues in its N-terminal half, each of which can contribute to the formation of cysteine-linked dimers. We previously reported that any one of the three cysteine residues is sufficient to produce functional BST-2 dimers. Here we investigated the importance of cysteine positioning on the ectodomain for functional dimerization of BST-2. Starting with a cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced at various locations throughout the ectodomain. The resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. We found significant flexibility in the positioning of cysteine residues, although the propensity to form cysteine-linked dimers generally decreased with increasing distance from the N terminus. Interestingly, all BST-2 variants, including the one lacking all three ectodomain cysteines, retained the ability to form non-covalent dimers, and all of the BST-2 variants were efficiently expressed at the cell surface. Importantly, not all BST-2 variants capable of forming cysteine-linked dimers were functional, suggesting that cysteine-linked dimerization of BST-2 is necessary but not sufficient for inhibiting virus release. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell.
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http://dx.doi.org/10.1074/jbc.M114.617639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319038PMC
February 2015

New insights into ubiquitin E3 ligase mechanism.

Nat Struct Mol Biol 2014 Apr;21(4):301-7

1] Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. [2] Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine. Three distinct classes of E3 ligases have been identified that stimulate transfer of ubiquitin and ubiquitin-like proteins through either a direct or an indirect mechanism. Only recently have the catalytic mechanisms of E3 ligases begun to be elucidated.
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http://dx.doi.org/10.1038/nsmb.2780DOI Listing
April 2014

Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase.

J Biol Chem 2013 Nov 17;288(45):32211-32228. Epub 2013 Sep 17.

From the Department of Pharmacology and Molecular Sciences,. Electronic address:

Ghrelin O-acyltransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the membrane-bound O-acyltransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, facilitated these experiments. The MBOAT family invariant residue His-338 is in the ER lumen, consistent with other family members, but conserved Asn-307 is cytosolic, making it unlikely that both are involved in catalysis. Photocross-linking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His-338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately advance the discovery of selective inhibitors for these enzymes.
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http://dx.doi.org/10.1074/jbc.M113.510313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820860PMC
November 2013

A conserved asparagine has a structural role in ubiquitin-conjugating enzymes.

Nat Chem Biol 2013 Mar 6;9(3):154-6. Epub 2013 Jan 6.

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

It is widely accepted that ubiquitin-conjugating enzymes contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13-Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop.
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http://dx.doi.org/10.1038/nchembio.1159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578109PMC
March 2013

RNF4-dependent hybrid SUMO-ubiquitin chains are signals for RAP80 and thereby mediate the recruitment of BRCA1 to sites of DNA damage.

Sci Signal 2012 Dec 4;5(253):ra88. Epub 2012 Dec 4.

Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.

The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double-strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. We demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin- and SUMO-dependent DSB recognition, revealing that RNF4-synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.
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http://dx.doi.org/10.1126/scisignal.2003485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131685PMC
December 2012

The size and conservation of a coiled-coil structure in the ectodomain of human BST-2/tetherin is dispensable for inhibition of HIV-1 virion release.

J Biol Chem 2012 Dec 14;287(53):44278-88. Epub 2012 Nov 14.

Laboratory of Molecular Microbiology, Viral Biochemistry Section, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460, USA.

BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.
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http://dx.doi.org/10.1074/jbc.M112.418822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531743PMC
December 2012

A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins.

Anal Biochem 2011 Nov 2;418(1):102-10. Epub 2011 Jul 2.

Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute and the Johns Hopkins University School of Medicine, Baltimore, MD 21202, USA.

Ubiquitination is a widely studied regulatory modification involved in protein degradation, DNA damage repair, and the immune response. Ubiquitin is conjugated to a substrate lysine in an enzymatic cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high-throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, and E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel-based assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.
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http://dx.doi.org/10.1016/j.ab.2011.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178097PMC
November 2011

Structural insights into the assembly and function of the SAGA deubiquitinating module.

Science 2010 May 15;328(5981):1025-9. Epub 2010 Apr 15.

Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

SAGA is a transcriptional coactivator complex that is conserved across eukaryotes and performs multiple functions during transcriptional activation and elongation. One role is deubiquitination of histone H2B, and this activity resides in a distinct subcomplex called the deubiquitinating module (DUBm), which contains the ubiquitin-specific protease Ubp8, bound to Sgf11, Sus1, and Sgf73. The deubiquitinating activity depends on the presence of all four DUBm proteins. We report here the 1.90 angstrom resolution crystal structure of the DUBm bound to ubiquitin aldehyde, as well as the 2.45 angstrom resolution structure of the uncomplexed DUBm. The structure reveals an arrangement of protein domains that gives rise to a highly interconnected complex, which is stabilized by eight structural zinc atoms that are critical for enzymatic activity. The structure suggests a model for how interactions with the other DUBm proteins activate Ubp8 and allows us to speculate about how the DUBm binds to monoubiquitinated histone H2B in nucleosomes.
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http://dx.doi.org/10.1126/science.1190049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220450PMC
May 2010

Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75.

Nat Struct Mol Biol 2008 Sep;15(9):948-56

Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 1300 University Avenue, Madison, Wisconsin 53706, USA.

Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by approximately 100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rttl09. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2678805PMC
http://dx.doi.org/10.1038/nsmb.1459DOI Listing
September 2008

Catalysis and substrate selection by histone/protein lysine acetyltransferases.

Curr Opin Struct Biol 2008 Dec;18(6):682-9

Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, 1300 University Avenue, Madison, WI 53706, USA.

Reversible protein acetylation is controlled by the opposing actions of protein lysine acetyltransferases and deacetylations. Recent developments on the structure and biochemical mechanisms of histone acetyltransferases (HATs) have provided new insight into catalysis and substrate selection. Diverse families of HATs appear to perform a conserved mechanism of acetyl transfer, where the lysine-containing substrate directly attacks enzyme-bound acetyl-CoA. The ability of HATs to form distinct multi-subunit complexes provides a means to regulate HAT activity by altering substrate specificity, targeting to specific loci, enhancing acetyltransferase activity, restricting access of non-target proteins, and coordinating the multiple enzyme activities of the complex. In the case of newly discovered Rtt109 HAT, association with distinct histone chaperones directs substrate selection between N-terminal lysines (H3K9, H3K23) and those (H3K56) within the histone fold domain. Moreover, the ability of some HATs to utilize longer chain acyl-CoA (i.e. propionyl-CoA) as alternative substrates suggests a potential direct link between the metabolic state of the cell and transcriptional regulation.
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http://dx.doi.org/10.1016/j.sbi.2008.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2723715PMC
December 2008

Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes.

Mol Cell 2007 Mar 22;25(5):703-12. Epub 2007 Feb 22.

Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation Street #506, Worcester, MA 01605, USA.

Acetylation of histone H3 on lysine 56 occurs during mitotic and meiotic S phase in fungal species. This acetylation blocks a direct electrostatic interaction between histone H3 and nucleosomal DNA, and the absence of this modification is associated with extreme sensitivity to genotoxic agents. We show here that H3-K56 acetylation is catalyzed when Rtt109, a protein that lacks significant homology to known acetyltransferases, forms an active complex with either of two histone binding proteins, Asf1 or Vps75. Rtt109 binds to both these cofactors, but not to histones alone, forming enzyme complexes with kinetic parameters similar to those of known histone acetyltransferase (HAT) enzymes. Therefore, H3-K56 acetylation is catalyzed by a previously unknown mechanism that requires a complex of two proteins: Rtt109 and a histone chaperone. Additionally, these complexes are functionally distinct, with the Rtt109/Asf1 complex, but not the Rtt109/Vps75 complex, being critical for resistance to genotoxic agents.
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http://dx.doi.org/10.1016/j.molcel.2007.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1853276PMC
March 2007

Nucleosome recognition by the Piccolo NuA4 histone acetyltransferase complex.

Biochemistry 2007 Feb 3;46(8):2091-9. Epub 2007 Feb 3.

Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

The mechanisms by which multisubunit histone acetyltransferase (HAT) complexes recognize and perform efficient acetylation on nucleosome substrates are largely unknown. Here, we use a variety of biochemical approaches and compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4 (picNuA4). We find the histone tails to be dispensable for binding to both nucleosomes and free histones and that the H2A, H3, and H2B tails do not influence the ability of picNuA4 to tetra-acetylate the H4 tail within the nucleosome. Most notably, we discovered that the histone-fold domain (HFD) regions of histones, particularly residues 21-52 of H4, are critical for tight binding and efficient tail acetylation. Presented evidence suggests that picNuA4 recognizes the open surface of the nucleosome on which the HFD of H4 is located. This binding mechanism serves to direct substrate access to the tails of H4 and H2A and allows the enzyme to be "tethered", thereby increasing the effective concentration of the histone tail and permitting successive cycles of H4 tail acetylation.
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http://dx.doi.org/10.1021/bi602366nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1994252PMC
February 2007

Catalytic mechanism of a MYST family histone acetyltransferase.

Biochemistry 2007 Jan;46(3):623-9

Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-epsilon-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate. Here, using the prototypical MYST family member Esa1, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 x 10(-4) s(-1), or approximately 2500-fold slower than histone acetylation; kcat = 1.6 s(-1)) and of CoA formation (0.0021 s(-1)) were inconsistent with a kinetically competent acetyl-enzyme intermediate. Previously, Cys-304 of Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (kcat) analysis of the wild type and C304A revealed an ionization (pKa = 7.6-7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced kcat approximately 200-fold at pH 7.5; however, at higher pH, E338Q exhibited nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-epsilon-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esa1 x acetyl-CoA x histone ternary complex.
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http://dx.doi.org/10.1021/bi602513xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752042PMC
January 2007