Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography

Fox, Brian; Yee, Vivien C.; Pedersen, Lars C.; Trong, I. Le; Bishop, Paul D.; Stenkamp, Ronald E.; Teller, David C.

doi:10.1074/jbc.274.8.4917

Cited by 110 publications

(132 citation statements)

References 47 publications

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“…The fXIIIa enzyme has one Ca 2ϩ ion at a location generally similar to site two of TGase 3 and lies in an acidic pocket formed by Asp 438 , Glu 485 , and Glu 490 , but the Ca 2ϩ ion coordinates only with the main chain carbonyl oxygen of Ala 457 , and four water molecules (34). However this ion does not confer any significant change in fXIIIa structure, and the enzyme is inactive.…”

Section: Resultsmentioning

confidence: 99%

Roles of Calcium Ions in the Activation and Activity of the Transglutaminase 3 Enzyme

Ahvazi

Boeshans

Idler

et al. 2003

Journal of Biological Chemistry

104

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The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca 2؉ ions, which in site one is constitutively bound, while those in sites two and three are acquired during activation and are required for activity. In particular, binding at site three opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction. In this study, we have solved the structures of three more forms of this enzyme by x-ray crystallography in the presence of Ca 2؉ and/or Mg 2؉ , which provide new insights on the precise contribution of each Ca 2؉ ion to activation and activity. First, we found that Ca 2؉ ion in site one can be exchanged with difficulty, and it has a binding affinity of K d ‫؍‬ 0.3 M (⌬H ‫؍‬ ؊6.70 ؎ 0.52 kcal/mol), which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca 2؉ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca 2؉ , or Mg 2؉ ; however, when Mg 2؉ is present, the enzyme is inactive, and the channel is closed. Thus Ca 2؉ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site. Transglutaminases (TGases)1 are ubiquitous enzymes that are used widely in biology for many different purposes. There are nine different genes for TGases in the human (1-5). Typically, TGases recognize and activate a protein-bound Gln residue by formation of a thiol-acyl intermediate form. The recognition of a Gln residue may be highly specific, such as is apparently the case of the factor XIIIa, TGase 3, and TGase 4 enzymes, or with rather low specificity as for the TGase 2 enzyme. Next, this acyl intermediate is approached by a nucleophilic second substrate, which transfers onto the Gln residue. Commonly, the nucleophile is water, resulting in the net deamidation of a target Gln residue. If the nucleophile is the ⑀-NH 2 of a protein-bound Lys residue, an isopeptide ⑀-(␥-glutamyl)lysine cross-link is formed. As this cannot be cleaved in animal cells, controlled TGase activity thereby provides an efficient way for the formation of stable, insoluble macromolecular complexes. Other nucleophiles used include polyamines (to form mono-or bi-substituted/cross-linked adducts) or -OH groups to form ester linkages (as in the case of the membranebound TGase 1 enzyme to link epidermis-specific ceramides required for barrier function) (6). The TGase 2 enzyme can bind GTP nucleotides, and there is a reciprocal relationship between this binding and transamidation reactivity (7-9), presumably because the GTP binds in the vicinity of where c...

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Section: Resultsmentioning

confidence: 99%

Roles of Calcium Ions in the Activation and Activity of the Transglutaminase 3 Enzyme

Ahvazi

Boeshans

Idler

et al. 2003

Journal of Biological Chemistry

104

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“…This ion (positioned analogously to the ion B magnesium in tmAC structures) serves as an anchoring point for ATP by coordinating and stabilizing the Pβ and γ-phosphate (Pγ) of the ATP analog. Soaking with SrCl 2 or EuCl 3 , which are both known to occupy calcium sites 18,19 , unambiguously identified this ion B site as a calcium-binding pocket (Fig. 1b).…”

mentioning

confidence: 93%

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

Steegborn

Litvin

Levin

et al. 2004

Nat Struct Mol Biol

154

240

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In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.The ubiquitous second messenger cAMP regulates a large variety of essential physiological processes such as gene expression, chromosome segregation and cellular metabolism. In mammalian cells, cAMP is synthesized by a family of nine transmembrane adenylyl cyclases (tmACs) and one sAC 1 . Unlike tmACs, which localize to the cellular membrane and respond to extracellular stimuli via heterotrimeric G proteins 1 , sAC is found in various intracellular compartments such as the mitochondria and the nucleus 2 . Its localization near intracellular cAMP targets is the impetus for current models of second messenger signal transduction, in which cAMP functions as a locally acting signaling molecule 2-4 .sAC is insensitive to the tmAC regulators calmodulin and heterotrimeric G proteins as well as the nonphysiological activator forskolin; instead, sAC senses physiological levels of bicarbonate 5 . Aside from its role as a universal physiological buffer maintaining cellular and extracellular pH, bicarbonate functions as a signaling molecule 3 , regulating many biological processes in mammals such as fertility 6 , acid-base homeostasis, breathing rate, metabolism and fluid transport (reviewed in ref. 7). As the only known signaling enzyme sensitive to physiological fluctuations of bicarbonate 5 , sAC probably mediates each of these processes. Bicarbonate activation of sAC is essential for sperm motility 8 as well as for pH-dependent COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. acid secretion in the epididymis and possibly the kidney 9 . In addition to its bicarbonate sensitivity, sAC is synergistically activated by calcium 10 , and this potentiation seems to be important for sperm maturation 11 . NIH Public AccessPrevious work has revealed the overall structure of tmAC enzymes and suggested a twometal ion mechanism for catalysis 12,13 . Despite their different regulation, mammalian sAC and tmACs are grouped into the nucleotidyl cyclase class III based on sequ...

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“…For example, the solved X-ray structures of the zymogen forms of hfXIIIa with or without a single Ca 2+ ion do not reveal any structural changes Yee et al, 1996;Fox et al, 1999). Furthermore, the structures of the fTG (Noguchi et al, 2001) and human TGase 2 (Liu et al, 2002) enzymes do not show any bound metal Ca 2+ ion.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, the X-ray structures indicate that the orientation of the His300/ Glu358 diad and charge distribution on the surface in TGase 3 could contribute specificity to the recognition of the K* substrate (Figures 6a, 7). This diad deprotonizes and correctly orients the K* side chain within the pocket for reaction with the thioacyl intermediate Pedersen et al, 1999). In particular, Trp327 of TGase 3 is located opposite Trp236 involved in oxyanion formation .…”

Section: Lysine Pocketmentioning

confidence: 99%

“…One of the most enigmatic aspects of catalysis by the TGases is that all are Ca 2+ ion dependent for both in vivo (Folk and Chung, 1985) and in vitro reactions (Fox et al, 1999;Ahvazi et al, 2002;Ahvazi et al, 2003), although few data are currently available on how or why their dependence on Ca 2+ metal ions is necessary. For example, the solved X-ray structures of the zymogen forms of hfXIIIa with or without a single Ca 2+ ion do not reveal any structural changes Yee et al, 1996;Fox et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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A model for the reaction mechanism of the transglutaminase 3 enzyme

Ahvazi

Steinert²

2003

Exp Mol Med

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A bstractTransglutam inase enzym es (TG ases) catalyze the calcium dependent formation of an isopeptide bond betw een protein-bound glutam ine and lysine substrates. Previously w e have show n that activated TG ase 3 acquires tw o additional calcium ions at site tw o and three. The calcium ion at site three results in the opening of a channel. A t this site, the channel opening and closing could m odulate, depending on which m etal is bound. Here we propose that the front of the channel could be used by the tw o substrates for enzym e reaction. W e propose that the glutam ine substrate is directed from Trp236 into the enzym e, shown by m olecular docking. Then a lysine substrate approaches the opened active site to engage Trp327, leading to form ation of the isopeptide bond. Further, direct com parisons of the structures of TG ase 3 w ith other TG ases have allow ed us to identify several residues that m ight potentially be involved in generic and specific recognition of the glutam ine and lysine substrates.Keywords: calcium ions; protein structure; residue specificity; TGase; TGase mechanism; X-ray crystallography IntroductionTransglutaminases (TGase; protein-glutamine: amine γ-glutamyl-transferase) are a family of calcium-dependent acyl-transfer enzymes that are widely expressed in biota (Folk and Chung, 1985;Greenberg et al., 1991;Melino et al., 1998;Melino et al., 2000;Fesus and Piacentini, 2002). Each of the eight active human TGase enzyme isoforms can activate a protein-bound Gln residue to form a thiol-acyl enzyme intermediate, which is attacked by a second nucleophilic substrate to accomplish the two-step reaction. The reaction leads to the formation of an isopeptide bond between two proteins and the covalent incorporation of polyamine into protein (Folk and Chung, 1985;Greenberg et al., 1991). The nucleophile may be: water, so that the activated Gln residue is deamidated to a Glu; a polyamine, so that a mono-substituted adduct or bi-substituted cross-link is formed; an alcohol, particularly the ω-hydroxyl group of certain long-chain ceramides expressed in mammalian epidermis, to form an ester; and perhaps most commonly, an ε-NH2 group of a protein bound Lys residue, resulting in the formation of an N ε -(γ-glutamyl)lysine isopeptide cross-link Nemes et al., 2000). This stabilizes macromolecular protein complexes. Typically, TGases recognize the generic sequence motif Gln-Gln*-Val for the first step of the reaction (where Gln* represents the targeted residue), although different isoforms display specificity as to which substrates bearing the motif may approach the enzyme . Anecdotal data suggest less specificity for Lys substrates (Tarcsa et al., 1998;Candi et al., 1999;.The three dimensional structures of four TGases have now been reported, i.e. human factor XIIIa (hfXIIIa) ), TGase 2 (Liu et al., 2002, TGase 3 enzymes (Ahvazi et al., 2002;2003), and a fish enzyme (fTG, equivalent to mammalian TGase 2, Noguchi et al., 2001). All consist of four domains that are similar in organization and fold (Figure 1...

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Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography

Cited by 110 publications

References 47 publications

Roles of Calcium Ions in the Activation and Activity of the Transglutaminase 3 Enzyme

Roles of Calcium Ions in the Activation and Activity of the Transglutaminase 3 Enzyme

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

A model for the reaction mechanism of the transglutaminase 3 enzyme

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