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PDBsum entry 1sgx

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protein ligands metals Protein-protein interface(s) links
Transferase PDB id
1sgx
Jmol
Contents
Protein chains
675 a.a. *
Ligands
5GP ×2
Metals
_CA ×4
_MG ×2
Waters ×861
* Residue conservation analysis
Obsolete entry
PDB id:
1sgx
Name: Transferase
Title: Crystal structure of transglutaminase 3 in complex with boun structural basis for alteration in nucleotide specificity
Structure: Protein-glutamine glutamyltransferase e. Chain: a, b. Synonym: tgase e, tge, tge, transglutaminase 3. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Tissue: foreskin. Gene: tgm3. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108. Expression_system_cell_line: sf9. (Invitrogen), bac-n-blue (invitrogen).
Resolution:
2.00Å     R-factor:   0.206     R-free:   0.241
Authors: B.Ahvazi
Key ref:
B.Ahvazi et al. (2004). Crystal structure of transglutaminase 3 in complex with GMP: structural basis for nucleotide specificity. J Biol Chem, 279, 26716-26725. PubMed id: 15084592 DOI: 10.1074/jbc.M403481200
Date:
24-Feb-04     Release date:   27-Apr-04    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
Q08188  (TGM3_HUMAN) -  Protein-glutamine gamma-glutamyltransferase E
Seq:
Struc:
 
Seq:
Struc:
693 a.a.
675 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 3 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.2.3.2.13  - Protein-glutamine gamma-glutamyltransferase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Protein glutamine + alkylamine = protein N5-alkylglutamine + NH3
Protein glutamine
+ alkylamine
= protein N(5)-alkylglutamine
+ NH(3)
      Cofactor: Ca(2+)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 

 
    reference    
 
 
DOI no: 10.1074/jbc.M403481200 J Biol Chem 279:26716-26725 (2004)
PubMed id: 15084592  
 
 
Crystal structure of transglutaminase 3 in complex with GMP: structural basis for nucleotide specificity.
B.Ahvazi, K.M.Boeshans, P.M.Steinert.
 
  ABSTRACT  
 
Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.
 
  Selected figure(s)  
 
Figure 2.
FIG. 2. A, left panels, stereo view of the 2.0-Å resolution 3 F[o] - 2 F[c] electron density map around the GMP contoured at 1.0- level in the. Right panel, view of cleft surface (yellow) and the surrounding residues within 6.5 Å of the GMP nucleotide located at the interface of the core domain (top) and the -barrel 1 domain (bottom). B, a view of the guanine nucleotide-binding site pocket in TGase 3·GMP complex. The hydrogen bonds and ion pair interactions are shown as dashed lines. All the important residues in the guanine nucleotide-binding site pocket and the GMP are shown in ball-and-stick. C, identification of key residues involved in the coordination with metal ions in sites 1, 2, and 3 in TGase 3 complex. In site 1, the Ca^2+ ion in the TGase 3·GMP forms direct contacts with the main chain carbonyl oxygen atoms of Ala^221, Asn224, Asn226, the carboxyl side-chain oxygen of Asn224, Asp 228, and to a water molecule. Site 2 exists near the end of catalytic core domain, next to the -helical segment (residues Asn430-Glu448), leading to the loop connecting the -barrel 1 domain. The Ca^2+ ion forms contacts with the carboxyl side-chain atoms of Asn393, Glu443, and Glu448, the main chain carbonyl oxygen atom of Ser415, and two water molecules. Mg2+ in site 3 binds in the vicinity of the loop segment, leading to the catalytic His330 at the active site. This Mg2+ ion forms contacts with the carboxyl side-chain oxygen atoms of Asp301 and Asp303, the side-chain atoms of Asn305, the main chain carbonyl oxygen atoms of Ser307, and a water molecule. The loop bearing residues 320DKGSDS325 has moved from its position in the native enzyme, closing the channel.
Figure 3.
FIG. 3. A, isothermal calorimetric titration of nucleotide binding to TGase 3. To solutions of the activated forms were added a series of injections of 5, 10, and 20 µl of GMP, and the heat absorbed or liberated was recorded (upper panel). The lower panel records the net heat changes. The heat of dilution was determined from different injections of a solution without protein subtracted to determine heat changes due solely to nucleotide binding. The data shown are representative of one of three experiments, each performed in triplicate; the S.D. of replicates was <5%. B, inhibition of TGase 3 by increasing concentrations of GMP for four TGase 3 concentrations ( , 1.0 mM; , 0.50 mM; , 0.25 mM; and , 0.10 mM). Increasing concentrations of GMP were preincubated for 30 min with recombinant activated TGase 3. C, activities of TGase 3 in the presence and absence of nucleotide were measured by incorporation of [14C]putrescine into casein. The - and + indicate the absence or presence of 0.575 mM Ca^2+ and 1 mM Mg2+ in the standard assay reaction.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 26716-26725) copyright 2004.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
18597041 A.Facchiano, and F.Facchiano (2009).
Transglutaminases and their substrates in biology and human diseases: 50 years of growing.
  Amino Acids, 36, 599-614.  
17024410 K.M.Boeshans, T.C.Mueser, and B.Ahvazi (2007).
A three-dimensional model of the human transglutaminase 1: insights into the understanding of lamellar ichthyosis.
  J Mol Model, 13, 233-246.  
17179049 G.E.Begg, L.Carrington, P.H.Stokes, J.M.Matthews, M.A.Wouters, A.Husain, L.Lorand, S.E.Iismaa, and R.M.Graham (2006).
Mechanism of allosteric regulation of transglutaminase 2 by GTP.
  Proc Natl Acad Sci U S A, 103, 19683-19688.  
15909997 B.F.Krasnikov, S.Y.Kim, S.J.McConoughey, H.Ryu, H.Xu, I.Stavrovskaya, S.E.Iismaa, B.M.Mearns, R.R.Ratan, J.P.Blass, G.E.Gibson, and A.J.Cooper (2005).
Transglutaminase activity is present in highly purified nonsynaptosomal mouse brain and liver mitochondria.
  Biochemistry, 44, 7830-7843.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time.