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

The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca2+ 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 Ca2+ and/or Mg2+, which provide new insights on the precise contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty, and it has a binding affinity of Kd = 0.3 μm (ΔH = –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 Ca2+ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca2+,orMg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ 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. The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca2+ 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 Ca2+ and/or Mg2+, which provide new insights on the precise contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty, and it has a binding affinity of Kd = 0.3 μm (ΔH = –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 Ca2+ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca2+,orMg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ 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) 1The abbreviations used are: TGase, transglutaminase; hfXIIIa, human factor XIIIa; NCS, non-crystallographic symmetry; PEG, polyethylene glycol; ICPMS-DRC, inductively coupled plasma-mass spectrometry dynamic reaction cell; r.m.s. root mean square. are ubiquitous enzymes that are used widely in biology for many different purposes. There are nine different genes for TGases in the human (1Folk J.E. Chung S.I. Methods Enzymol. 1985; 113: 358-375Google Scholar, 2Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Google Scholar, 3Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar, 4Melino G. Candi E. Steinert P.M. Methods Enzymol. 2000; 322: 433-472Google Scholar, 5Fesus L. Piacentini M. Trends Biochem. Sci. 2002; 27: 534-539Google Scholar). 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 ϵ-NH2 of a protein-bound Lys residue, an isopeptide ϵ-(γ-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 membrane-bound TGase 1 enzyme to link epidermis-specific ceramides required for barrier function) (6Nemes Z. Marekov L.N. Fesus L. Steinert P.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8402-8407Google Scholar). The TGase 2 enzyme can bind GTP nucleotides, and there is a reciprocal relationship between this binding and transamidation reactivity (7Lee K.N. Birckbichler P.J. Patterson Jr., M.K. Biochem. Biophys. Res. Commun. 1989; 162: 1370-1375Google Scholar, 8Im M.J. Graham R.M. J. Biol. Chem. 1990; 265: 18944-18951Google Scholar, 9Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.J. Graham R.M. Science. 1994; 264: 1593-1596Google Scholar), presumably because the GTP binds in the vicinity of where cross-linking substrates must gain access to the enzyme for reaction (10Liu S. Cerione R.A. Clardy J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2743-2747Google Scholar). It remains to be demonstrated whether other TGase isoforms can also manipulate nucleotides. In addition, TGase enzyme transamidation reactions require Ca2+, in both in vitro assays and in vivo (1Folk J.E. Chung S.I. Methods Enzymol. 1985; 113: 358-375Google Scholar, 2Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Google Scholar, 3Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar, 4Melino G. Candi E. Steinert P.M. Methods Enzymol. 2000; 322: 433-472Google Scholar, 5Fesus L. Piacentini M. Trends Biochem. Sci. 2002; 27: 534-539Google Scholar). In the few cases measured, the Ca2+ concentration required to activate an enzyme isoform (>500 μm) is far higher than net intracellular Ca2+ ion concentrations (about 100 nm) (11Nemes Z. Marekov L.N. Steinert P.M. J. Biol. Chem. 1999; 274: 11013-11021Google Scholar). Also, Ca2+ is not required for GTP binding (7Lee K.N. Birckbichler P.J. Patterson Jr., M.K. Biochem. Biophys. Res. Commun. 1989; 162: 1370-1375Google Scholar, 8Im M.J. Graham R.M. J. Biol. Chem. 1990; 265: 18944-18951Google Scholar, 9Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.J. Graham R.M. Science. 1994; 264: 1593-1596Google Scholar). Thus manipulation of intracellular Ca2+ concentrations could afford an effective way to control TGase functions, including cross-linking. Despite the evident essential role of Ca2+ ions in TGase cross-linking reactions, very little is known structurally/functionally why Ca2+ ions are in fact required. Recently however, we solved the structure of the zymogen and the activated form of the TGase 3 enzyme system (12Kim H.C. Nemes Z. Idler W.W. Hyde C.C. Steinert P.M. Ahvazi B. J. Struct. Biol. 2001; 135: 73-77Google Scholar, 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). Whereas the zymogen constitutively acquires one ion during expression (in baculovirus), it is insufficient for activity. Upon proteolytic cleavage of a loop segment connecting the active site domain to the β-barrel 1 domain, the cleaved enzyme can acquire two additional Ca2+ ions, and becomes fully active. These events coincide with the opening of a channel, which passes through the enzyme. Moreover, this channel exposes two tryptophan residues, which are believed to be important in the enzyme reaction mechanism (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). The channel is formed upon the opening of an existing deep cavity by movement of a β-strand loop (residues Gly322–Ser325) so that Asp324 can coordinate with the Ca2+ ion that occupies the site nominally termed site three. We also showed that certain trivalent lanthanide ions can occupy sites three and/or two with retention of activity and an open channel. However, key questions remain unanswered. What are the relative contributions of the occupancy of each Ca2+ ion in the two sites, and why is there a site two? Does the proteolyzed form in the absence of metal ions in sites two and three possess a channel, or partially opened channel, or no channel? Nor of course are lanthanide ions physiologic. Accordingly, here we present the structures of three more forms of the proteolyzed TGase 3 by x-ray crystallography crystallized in the absence or presence of Ca2+ and/or more biologically relevant Mg2+ new data that Ca2+ ions are essential in sites two and three to open the channel for and that ion in site three cannot open the channel to activate the enzyme. that the intracellular concentration of Mg2+ ions is of higher than for Ca2+ ions, these data that the opening of a channel and of activity can be controlled by the and by of metal Moreover, these data that this channel is required for enzyme and human TGase 3 zymogen expressed in the system and as (12Kim H.C. Nemes Z. Idler W.W. Hyde C.C. Steinert P.M. Ahvazi B. J. Struct. Biol. 2001; 135: 73-77Google Scholar). the proteolyzed form a of zymogen in a of 1 and with 3 of at for However, the of Ca2+ Accordingly, to the proteolyzed form of Ca2+ ions, through a with a of and at (12Kim H.C. Nemes Z. Idler W.W. Hyde C.C. Steinert P.M. Ahvazi B. J. Struct. Biol. 2001; 135: 73-77Google Scholar). is here as form we found that the of and 1 to form in the form which a Ca2+ ion in site two and a Mg2+ ion in site the not We to in which only site two or site three an and data proteolyzed with for proteolyzed with activation and proteolyzed with and with and = = = = = = = = = = = = = = of of of is of of = where are the and structure factor for of the data proteolyzed with for proteolyzed with activation and proteolyzed with and with and In is = where are the and structure factor for of the data in a new of form the by the to 100 of in 1 and at a concentration of a 2 of 2 of and a and 100 at The of TGase 3 form in 100 of in 1 and a 4 of 2 of and a or 100 at The TGase 3 form in 100 of in 1 and 1 a 4 of 2 of and a 100 100 at as the and with a loop to data data at the the 4 with a of of and of and for The data and the of Z. Methods Enzymol. Scholar). to data in which ions are as in the during a Z. M. 2000; Scholar). The in 1 for but no could be The data of are in and structure of the TGase 3 proteolyzed form solved by J. Methods Enzymol. and a of to for the and between to for the There two to both the and These to two each in the form in The of the TGase enzyme 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google used as the The and the two for the data to The the J. M. Rice T. Scholar), by non-crystallographic to the a of 1994; with 5: Scholar). the many of 3 the at an of which low data as in the on and the presence of in on on the In to of the each of the the and a residue as a the TGase 3 data with one of the of the zymogen form 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google as the in J. Methods Enzymol. used to for the of in this form in the = The in J. M. Rice T. with the and a target A. A. 1990; Scholar). and a and the for the of of the data data between and no in the which at an of The structure of the TGase 3 form solved by J. Methods Enzymol. Scholar). There two to both the and These to two each in the The of the TGase enzyme 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google used as the The J. M. Rice T. by 5: Scholar). the many of 3 and the at an of which low data as In each of by a which used to or the The of the structure with the R.A. J. and J. M. Rice T. Scholar). with P.J. J. 1991; and Methods Enzymol. Scholar). of TGase of Ca2+ binding to the activated TGase 3 by a as previously (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). to binding and of metal ion by the of inductively coupled plasma-mass spectrometry dynamic reaction a to the TGase 3 with at for and and The by The concentration of zymogen with TGase 3 activity by of J.E. Biophys. Scholar). at in of 100 of 1 and 1 TGase 3 Ca2+ binding by of the enzyme 100 of with of to at 4 of the enzyme and of the for The to the concentration by at The of this is to the of in the three known of the TGase 3 enzyme system and to in the activation We have solved the structures of three additional and are in of data on of in TGase 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google proteolyzed with ± on ± 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google ± in a new the structure of TGase 3 of that are to the structures solved here and previously (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). The domain of TGase 3 of nine of with three two with to each other to form a The domain (residues of with and is It a which two of The of residues is in the of the and the active site residue, which is in this Other of the and are on of The 1 and 2 of of in residues and to form a highly loop that the segment of the domain to the β-strand of the 1 the cleavage site used for proteolytic activation of the TGase 3 structures possess two in the to the loop of that the active site and in a loop two of the the is in in The of to the TGase we to whether the channel is opened by cleavage of the enzyme that of metal ions at sites two and three. of the TGase 3 zymogen with at the of that the active site domain to β-barrel 1 form in the presence of but with no Ca2+, is fully active x-ray structure is shown in The are in The x-ray of two of have for a loop between residues The r.m.s. between the of the two is that the structures are has three Ca2+ ions, and an open channel However, its structure and of the Ca2+ ions form and are to those of the activated solved previously 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google and of It that the used of Ca2+ so that during the cleaved enzyme acquires the two additional Ca2+ ions the to occupy sites two and three. However, the of the two in the of the structure is the different the previously (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). for between the in the a of for the upon formation of the two of key residues in the with metal ions in sites and three in forms The of these and the are in of residues 3 of the ion in a new the Ca2+ at and to the two Ca2+ ions by through a by of and this form It has one of residues with for residues in its of the and enzyme is inactive, and the channel is of its structures and the of its Ca2+ ion at site one near to the zymogen form and and 13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). The only is that the loop has so that and coordinate with the Ca2+ ion in site are with the Ca2+ thereby binding the ion more In these data that metal ions must occupy sites two and/or three to open the channel for activity. The Ca2+ in Site to TGase we to the of binding a Ca2+ ion at site However, by of more we to of this as by with and of the binding data for the Ca2+ ions 3, and the Ca2+ ion there is one affinity binding site Kd = 0.3 μm) with = –6.70 ± 0.52 of Ca2+ two other low affinity sites Kd = μm) with = ± The of the of the and are with the data for Ca2+ ion binding at sites two and three (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). Thus the of the to binding at site an reaction stabilization of the and we showed that lanthanides can occupy sites two and/or three with retention of activity (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). the metal cation in is Accordingly, we to occupy sites two or three with Ca2+ or Mg2+ First, we TGase 3 activity in the presence of and/or there only of activity. activity acquired by in the absence and presence of of higher concentrations of activity by we form with different of these ions and found by that in the of or at low of the TGase 3 one of each ion only acquired a second Ca2+ ion by to the Mg2+ ion by and Mg2+ ion by The one Mg2+ enzyme no the two Mg2+ enzyme very low and the three Ca2+ enzyme activity to form with one Mg2+, and one of and an because of Mg2+, and the not bind to TGases K. K. M. Biochem. 2000; Scholar), we to only the second of termed form We by in the presence of one Mg2+ and two Ca2+ ions and no other The x-ray structure of form has two of residues at in the and have for a loop between residues and and the r.m.s. between the of the two is that the structures are for between the in the a of for the upon formation each As the is a in H.C. J.E. J.E. Chung S.I. J. Biol. Chem. 1990; 265: Scholar), this may not be relevant and is by the the channel is and There are in the of the metal ions form and while sites one and two are as in form site three is with only in an its and not the residue which of sites two or three the Mg2+ we both for of and a in the factor for Ca2+ relative to the and to that site three the Mg2+ we the mean in the at for each metal ion in sites two and three in each and found that site two is Ca2+ and site three The of the the and the the of Mg2+ at site three. as as and M. B. 1991; for this as Mg2+ We that the to the for Mg2+ and for In addition, the of Mg2+ ions a for rather than an more for Ca2+ A. J. A. Scholar). only the of is in a to Mg2+, Ca2+ in a with both the of in form that a Ca2+ ion at site three. Thus the residue an important role in between Ca2+ and Mg2+ of the Ca2+ and Mg2+ of forms and that the of the metal ions are however, of structures that the Mg2+ ion at site three in a of the and a because of a with Moreover, the Mg2+ ion in site three is to coordinate with residue Asp324 the loop segment Thus this loop occupies the as in the zymogen and no channel the other some lanthanide metal ion or forms as active as the Ca2+ form (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar). The is that for Ca2+ and both possess and are of effective for are and In the in metal ion binding at site 3, we that a the residue a it provides a of than other a the of or of this by thereby of Asp324 with a Ca2+ could the affinity and of metal ion binding or and control the to between metal ions of different as here for Ca2+ and Mg2+ there are for this Scholar, J. K. M. Biol. 2001; Scholar, D.M. H.C. H. K. H. Scholar). In particular, on has that residues can the and/or of metal binding Scholar). Accordingly, these data that the metal binding of site three the precise with which in the of the of the Ca2+ in Site 4 showed that in the presence of relative concentrations of Mg2+ ions, the proteolyzed enzyme the one Ca2+ ion at site one and a Mg2+ and the enzyme is The data for form that the Mg2+ ion be at site 3 site two As relative Ca2+ ion concentrations the enzyme acquires a second ion but the two Mg2+ ion form is data that this second Ca2+ ion occupies site However, as relative Ca2+ ion concentrations the Mg2+ ion at site three is by Ca2+, and only when a net of Ca2+ ions have the enzyme to activity We this to opening of the of the enzyme. The of the activity of suggests that site two be with the Ca2+ ion and that activity is as as site three is at These data that sites two and three cooperate with each We the why site two is not occupied by a Mg2+ ion in a Mg2+ ion of 2 and suggests that in to with the a Mg2+ ion could only coordinate with one at a The of Mg2+ ion its for a of with an of its and is than Ca2+ in and for a Mg2+ ion to coordinate with of the are site two provides a highly binding cavity for metal ions, so that only a Ca2+ ion with an and can occupy site The enzyme has one Ca2+ ion at a to site two of TGase 3 and in an formed by and but the Ca2+ ion only with the of and J. Biol. Chem. 1999; 274: Scholar). this ion not in and the enzyme is It remains to be whether other Ca2+ ions are by an active enzyme form and whether cooperate with each other to reaction. of the the structure of the zymogen (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google and form the presence of a deep cavity on one termed the of the TGase 3 enzyme. We have in form that the binding of the Ca2+ ion in site three a loop the residues to so that Asp324 with the ion loop on the of the enzyme so as to open the deep cavity and a channel through the enzyme. However, when the Ca2+ ion is by a Mg2+ ion at this site the channel is and The of in the loop forms a a to the of and the of the of the to be closed. the of the the cavity is open whether or not metal ion occupies site three. The of near the opening of the cavity to the is the of the active site and the of The of the cavity is the of to the metal ion at site three. its the of the cavity is the of to the of with a of The of the cavity is controlled by the of the domain and of the β-strand of the β-barrel 1 domain These residues to be with each other and in The of the cavity is by of and and is with a of to each other through The cavity is by the of and and the of the the groups of three in the and at the of the the channel. The of the cavity not in the structures solved here or previously (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar), for the opening of the these data on this could be used by it is deep for a Gln or Lys residue to to the active site residue and it is for a a Gln or Lys residue to through the of to the active site residue. Accordingly, it is to the cavity at the of the enzyme could be for substrates the enzyme the of the of by the TGase 3 enzyme is its on two metal occupy binding sites and in for metal The concentration of Mg2+ ions in is that for Ca2+ ions is so that there is a the for the control of of the TGase 3 enzyme. First, TGase 3 present in a be in the zymogen form and have a Ca2+ ion in site the loop of residues of TGase 3 is to activation (13Ahvazi B. Kim H.C. Kee S.H. Nemes Z. Steinert P.M. EMBO J. 2002; 21: 2055-2067Google Scholar, H.C. J.E. J.E. Chung S.I. J. Biol. Chem. 1990; 265: Scholar). enzyme proteolyzed or we can the data of 4 that site three acquire a Mg2+ ion but the enzyme form remain provides a but mechanism to the transamidation reactions by some proteolyzed TGase 3 enzyme. when intracellular or Ca2+ concentrations the site two acquire a Ca2+ and the Mg2+ ion site three be by a Ca2+ ion The Ca2+ ion occupancy at both sites with residues and resulting in a in the of the loop the channel and the enzyme becomes active. can Ca2+ concentrations to the required in We speculate that Ca2+ ion could be in by of the TGase 3 enzyme with other as has demonstrated previously for the TGase 1 enzyme Z. Marekov L.N. Steinert P.M. J. Biol. Chem. 1999; 264: 11013-11021Google Scholar). are to this channel is by substrates to reaction.

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