Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1

2015 ◽  
Vol 467 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Satoru Yuzawa ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Catalytic Domain ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Stable Complex ◽  
Amino Acid Residues ◽  
Substrate Selection ◽  
Post Translational Modification ◽  
Acetyl Group

The functions of microtubules are controlled in part by tubulin post-translational modification including acetylation of Lys40 in α-tubulin. αTAT1 (α-tubulin acetyltransferase 1), an enzyme evolutionarily conserved among eukaryotes, has recently been identified as the major α-tubulin Lys40 acetyltransferase, in which AcCoA (acetyl-CoA) serves as an acetyl group donor. The regulation and substrate recognition of this enzyme, however, have not been fully understood. In the present study, we show that AcCoA and CoA each form a stable complex with human αTAT1 to maintain the protein integrity both in vivo and in vitro. The invariant residues Arg132 and Ser160 in αTAT1 participate in the stable interaction not only with AcCoA but also with CoA, which is supported by analysis of the present crystal structures of the αTAT1 catalytic domain in complex with CoA. Alanine substitution for Arg132 or Ser160 leads to a drastic misfolding of the isolated αTAT1 catalytic domain in the absence of CoA and AcCoA but not in the presence of excess amounts of either cofactor. A mutant αTAT1 carrying the R132A or S160A substitution is degraded much faster than the wild-type protein when expressed in mammalian Madin–Darby canine kidney cells. Furthermore, alanine-scanning experiments using Lys40-containing peptides reveal that α-tubulin Ser38 is crucial for substrate recognition of αTAT1, whereas Asp39, Ile42, the glycine stretch (amino acid residues 43–45) and Asp46 are also involved. The requirement for substrate selection is totally different from that in various histone acetyltransferases, which appears to be consistent with the inability of αTAT1 to acetylate histones.

Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1

2001 ◽  
Vol 361 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Xinguo QIAN ◽  
Wenbo HOU ◽  
Li ZHENGANG ◽  
Bingdong SHA
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Peptide Binding ◽  
Stable Complex ◽  
Amino Acid Residues ◽  
Docking Model ◽  
Upper Level ◽  
Lid Domain

Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge–charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1–Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an ‘anchoring and docking’ model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.

scholarly journals Structural basis for acyl-group discrimination by human Gcn5L2

2016 ◽  
Vol 72 (7) ◽  
pp. 841-848 ◽  
Author(s):  
Alison E. Ringel ◽  
Cynthia Wolberger
Keyword(s):  
Catalytic Domain ◽  
Acyl Chain ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Lysine Acetylation ◽  
Acyltransferase Activity ◽  
Acyl Chain Length ◽  
Acetyl Group ◽  
Weak Activity

Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modificationsin vivo, the acyl-chain specificity of the acetyltransferase human Gcn5 (Gcn5L2) was examined. Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 becomes progressively weaker with increasing acyl-chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA were determined. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor of Gcn5L2versusacetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl-chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group.

scholarly journals The regulatory domain of protein kinase C-ε restricts the catalytic-domain-specificity

1991 ◽  
Vol 276 (1) ◽  
pp. 257-260 ◽  
Author(s):  
C Pears ◽  
D Schaap ◽  
P J Parker
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Structural Basis ◽  
Regulatory Domain ◽  
Substrate Selection ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Protein kinase C (PKC) consists of a family of closely related enzymes that can be divided into two subfamilies (alpha, beta and gamma and delta, epsilon and zeta) on the basis of primary sequence. Functional differences have also been described; thus PKC-alpha, PKC-beta and PKC-gamma readily phosphorylate histone IIIS in vitro, whereas PKC-epsilon will not employ this substrate efficiently. We have previously demonstrated, however, that proteolytic cleavage of PKC-epsilon generates a constitutive kinase activity that is an efficient histone IIIS kinase [Schaap, Hsuan, Totty & Parker (1990) Eur. J. Biochem. 191, 431-435]. In order to investigate the structural basis for this switch in specificity, we have constructed a chimaeric protein containing the regulatory domain of PKC-epsilon fused to the catalytic domain of PKC-gamma. When this is expressed in COS1 cells the chimaeric kinase shows a substrate-specificity similar to that of PKC-epsilon rather than to that of PKC-gamma. This demonstrates a role for the regulatory domain in substrate selection of PKC-epsilon.

scholarly journals Bacterial O-GlcNAcase genes abundance decreases in ulcerative colitis patients and its administration ameliorates colitis in mice

Gut ◽  
2020 ◽  
pp. gutjnl-2020-322468
Author(s):  
Xiaolong He ◽  
Jie Gao ◽  
Liang Peng ◽  
Tongtong Hu ◽  
Yu Wan ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gut Microbiota ◽  
Catalytic Domain ◽  
Host Cells ◽  
In Vivo Studies ◽  
Post Translational Modification ◽  
Human Gut ◽  
Colonic Inflammation

ObjectiveO-linked N-acetylglucosaminylation (O-GlcNAcylation), controlled by O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), is an important post-translational modification of eukaryotic proteins and plays an essential role in regulating gut inflammation. Gut microbiota encode various enzymes involved in O-GlcNAcylation. However, the characteristics, abundance and function of these enzymes are unknown.DesignWe first investigated the structure and taxonomic distribution of bacterial OGAs and OGTs. Then, we performed metagenomic analysis to explore the OGA genes abundance in health samples and different diseases. Finally, we employed in vitro and in vivo experiments to determine the effects and mechanisms of bacterial OGAs to hydrolyse O-GlcNAcylated proteins in host cells and suppress inflammatory response in the gut.ResultsWe found OGAs, instead of OGTs, are enriched in Bacteroidetes and Firmicutes, the major bacterial divisions in the human gut. Most bacterial OGAs are secreted enzymes with the same conserved catalytic domain as human OGAs. A pooled analysis on 1999 metagenomic samples encompassed six diseases revealed that bacterial OGA genes were conserved in healthy human gut with high abundance, and reduced exclusively in ulcerative colitis. In vitro studies showed that bacterial OGAs could hydrolyse O-GlcNAcylated proteins in host cells, including O-GlcNAcylated NF-κB-p65 subunit, which is important for activating NF-κB signalling. In vivo studies demonstrated that gut bacteria-derived OGAs could protect mice from chemically induced colonic inflammation through hydrolysing O-GlcNAcylated proteins.ConclusionOur results reveal a previously unrecognised enzymatic activity by which gut microbiota influence intestinal physiology and highlight bacterial OGAs as a promising therapeutic strategy in colonic inflammation.

scholarly journals Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

1992 ◽  
Vol 12 (10) ◽  
pp. 4634-4642 ◽  
Author(s):  
B Rubinfeld ◽  
W J Crosier ◽  
I Albert ◽  
L Conroy ◽  
R Clark ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Gtpase Activating Protein ◽  
Mutant Proteins ◽  
Carboxy Terminal

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

scholarly journals Structural Basis of Stu2 Recruitment to Yeast Kinetochores

2020 ◽  
Author(s):  
Jacob A. Zahm ◽  
Michael G. Stewart ◽  
Joseph S. Carrier ◽  
Stephen C. Harrison ◽  
Matthew P. Miller
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Assembly Checkpoint ◽  
Terminal Segment ◽  
Structural Basis ◽  
Amino Acid Residues ◽  
Checkpoint Signaling ◽  
Delay Cell ◽  
Sister Chromatids

ABSTRACTAccurate chromosome segregation during cell division requires engagement of the kinetochores of sister chromatids with microtubules emanating from opposite poles of the mitotic spindle. In yeast, these “bioriented” metaphase sister chromatids experience tension as the corresponding microtubules (one per sister chromatid) shorten. Spindle-assembly checkpoint signaling appears to cease from a kinetochore under tension, which also stabilizes kinetochore-microtubule attachment in single-kinetochore experiments in vitro. The microtubule polymerase, Stu2, the yeast member of the XMAP215/ch-TOG protein family, associates with kinetochores in cells and contributes to tension-dependent stabilization, both in vitro and in vivo. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that amino-acid residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell-cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function. These findings show that the role of Stu2 in tension sensing depends on its association with kinetochores by binding with Ndc80c.

scholarly journals Structural basis for acyl group discrimination by human Gcn5L2

2016 ◽  
Author(s):  
Alison E. Ringel ◽  
Cynthia Wolberger
Keyword(s):  
Catalytic Domain ◽  
Acyl Chain ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Lysine Acetylation ◽  
Acyltransferase Activity ◽  
Acyl Chain Length ◽  
Acetyl Group ◽  
Weak Activity

Abstract Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modifications in vivo, we examined the acyl chain specificity of the acetyltransferase, human Gcn5 (Gcn5L2). Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 gets progressively weaker with increasing acyl chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, we determined structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor for Gcn5L2 versus acetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group.

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis

1992 ◽  
Vol 12 (10) ◽  
pp. 4634-4642
Author(s):  
B Rubinfeld ◽  
W J Crosier ◽  
I Albert ◽  
L Conroy ◽  
R Clark ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Gtpase Activating Protein ◽  
Mutant Proteins ◽  
Carboxy Terminal

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.

scholarly journals Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity

2021 ◽  
Vol 22 (21) ◽  
pp. 11480
Author(s):  
Jiachi Chiou ◽  
Qipeng Cheng ◽  
Perry Tim-fat Shum ◽  
Marcus Ho-yin Wong ◽  
Edward Wai-chi Chan ◽  
...  
Keyword(s):  
Substrate Recognition ◽  
Functional Characterization ◽  
Salt Bridge ◽  
Structural Basis ◽  
Active Site Residues ◽  
Carboxylate Oxygen ◽  
Class D ◽  
Hydrolysis Of

Class D β-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this β-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various β-lactams were modeled and the potential active site residues that may interact with various β-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the β-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type β-lactamases.

scholarly journals Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0257972
Author(s):  
Hassan Issafras ◽  
Shilong Fan ◽  
Chi-Ling Tseng ◽  
Yunchih Cheng ◽  
Peihua Lin ◽  
...  
Keyword(s):  
Cancer Immunotherapy ◽  
Progression Free Survival ◽  
Structural Basis ◽  
Amino Acid Residues ◽  
Functional Activities ◽  
Human T Cells ◽  
Cancer Immunotherapies ◽  
Anti Tumor Activity

Cancer immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), represents a breakthrough in cancer treatment, resulting in unprecedented results in terms of overall and progression-free survival. Discovery and development of novel anti PD-1 inhibitors remains a field of intense investigation, where novel monoclonal antibodies (mAbs) and novel antibody formats (e.g., novel isotype, bispecific mAb and low-molecular-weight compounds) are major source of future therapeutic candidates. HLX10, a fully humanized IgG4 monoclonal antibody against PD-1 receptor, increased functional activities of human T-cells and showed in vitro, and anti-tumor activity in several tumor models. The combined inhibition of PD-1/PDL-1 and angiogenesis pathways using anti-VEGF antibody may enhance a sustained suppression of cancer-related angiogenesis and tumor elimination. To elucidate HLX10’s mode of action, we solved the structure of HLX10 in complex with PD-1 receptor. Detailed epitope analysis showed that HLX10 has a unique mode of recognition compared to the clinically approved PD1 antibodies Pembrolizumab and Nivolumab. Notably, HLX10’s epitope was closer to Pembrolizumab’s epitope than Nivolumab’s epitope. However, HLX10 and Pembrolizumab showed an opposite heavy chain (HC) and light chain (LC) usage, which recognizes several overlapping amino acid residues on PD-1. We compared HLX10 to Nivolumab and Pembrolizumab and it showed similar or better bioactivity in vitro and in vivo, providing a rationale for clinical evaluation in cancer immunotherapy.

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