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Structural Requirements for N-Trimethylation of Lysine 115 of Calmodulin

2000; Elsevier BV; Volume: 275; Issue: 25 Linguagem: Inglês

10.1074/jbc.m002332200

1083-351X

Jennifer A. Cobb, Daniel M. Roberts,

Ammonia Synthesis and Nitrogen Reduction

Calmodulin is trimethylated at lysine 115 by a highly specific methyltransferase that utilizesS-adenosylmethionine as a co-substrate. Lysine 115 is found within a highly conserved six-amino acid loop (LGEKLT) that forms a 90° turn between EF-hand III and EF-hand IV in the carboxyl-terminal lobe. In the present work a mutagenesis approach was used to investigate the structural features of the carboxyl-terminal lobe that lead to the specificity of calmodulin methylation. Three structural regions within the carboxyl-terminal lobe appear to be involved in methyltransferase recognition: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent α-helices (helix 6 and helix 7) from EF-hands III and IV. Site-directed mutagenesis of residues in the loop show that three residues, glycine 113, glutamate 114, and leucine 116 are essential for methylation. In addition, subdomain (individual helix or Ca2+ binding loop) exchange mutants show that the substitutions of either helix 6 (EF-hand III) with helix 2 (EF-hand I) or helix 7 (EF-hand IV) with helix 3 (EF-hand II) compromises methylation. Charge-to-alanine mutations in helix 7 show that substitution of conserved charged residues at positions 118, 120, 122, 126, and 127 reduced lysine 115 methylation rates, suggesting possible electrostatic interactions between this helix and the methyltransferase. Single substitutions in helix 6 did not affect calmodulin methylation, suggesting this region may play a more indirect role in stabilizing the conformation of the methyltransferase recognition sequence. Calmodulin is trimethylated at lysine 115 by a highly specific methyltransferase that utilizesS-adenosylmethionine as a co-substrate. Lysine 115 is found within a highly conserved six-amino acid loop (LGEKLT) that forms a 90° turn between EF-hand III and EF-hand IV in the carboxyl-terminal lobe. In the present work a mutagenesis approach was used to investigate the structural features of the carboxyl-terminal lobe that lead to the specificity of calmodulin methylation. Three structural regions within the carboxyl-terminal lobe appear to be involved in methyltransferase recognition: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent α-helices (helix 6 and helix 7) from EF-hands III and IV. Site-directed mutagenesis of residues in the loop show that three residues, glycine 113, glutamate 114, and leucine 116 are essential for methylation. In addition, subdomain (individual helix or Ca2+ binding loop) exchange mutants show that the substitutions of either helix 6 (EF-hand III) with helix 2 (EF-hand I) or helix 7 (EF-hand IV) with helix 3 (EF-hand II) compromises methylation. Charge-to-alanine mutations in helix 7 show that substitution of conserved charged residues at positions 118, 120, 122, 126, and 127 reduced lysine 115 methylation rates, suggesting possible electrostatic interactions between this helix and the methyltransferase. Single substitutions in helix 6 did not affect calmodulin methylation, suggesting this region may play a more indirect role in stabilizing the conformation of the methyltransferase recognition sequence. Calmodulin is a highly conserved calcium sensor protein that modulates the activities of multiple enzymes. Calmodulin is a monomer consisting of two structurally similar globular calcium binding lobes (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar) connected by a flexible linker region (2.Persechini A. Kretsinger R.H. J. Biol. Chem. 1988; 263: 12175-12178Abstract Full Text PDF PubMed Google Scholar, 3.Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (887) Google Scholar). Each lobe consists of two helix-loop-helix EF-hand calcium binding sites, with EF-hand domains I and II constituting the amino-terminal lobe and EF-hands III and IV constituting the carboxyl-terminal lobe. Many naturally occurring calmodulins are posttranslationally trimethylated on a single lysine residue at position 115 (reviewed in Ref. 4.Siegel F.L. Vincent P.L. Neal T.L. Wright L.S. Heth A.A. Rowe P.M. Paik W.K. Kim S. Protein Methylation. CRC Press, Inc., Boca Raton, FL1990: 33-58Google Scholar). Lysine 115 is a solvent-exposed residue that is found on a highly conserved six-amino acid loop-turn region (LGEKLT) located between helix 6 of EF-hand III and helix 7 of EF-hand IV (Fig. 1). Trimethylation of calmodulin at lysine 115 is catalyzed by an N-methyltransferase that utilizes S-adenosylmethionine as a co-substrate (Refs. 5.Rowe P.M. Wright L.S. Siegel F.L. J. Biol. Chem. 1986; 261: 7060-7069Abstract Full Text PDF PubMed Google Scholar, 6.Morino H. Kawamoto T. Miyake M. Kakimoto Y. J. Neurochem. 1987; 48: 1201-1208Crossref PubMed Scopus (18) Google Scholar, 7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar, 8.Pech L.L. Nelson D.L. Biochim. Biophys. Acta. 1994; 1199: 183-194Crossref PubMed Scopus (8) Google Scholar, 9.Wright L.S. Bertics P.J. Siegel F.L. J. Biol. Chem. 1996; 271: 12737-12743Abstract Full Text PDF PubMed Scopus (11) Google Scholar, reviewed in Ref. 4.Siegel F.L. Vincent P.L. Neal T.L. Wright L.S. Heth A.A. Rowe P.M. Paik W.K. Kim S. Protein Methylation. CRC Press, Inc., Boca Raton, FL1990: 33-58Google Scholar). This enzyme appears to have the dedicated function of trimethylating lysine 115 in calmodulin from a wide variety of species. From a functional perspective, calmodulin methylation selectively affects the regulation of certain enzymes such as NAD kinase (10.Roberts D.M. Rowe P.M. Siegel F.L. Lukas T.J. Watterson D.M. J. Biol. Chem. 1986; 261: 1491-1494Abstract Full Text PDF PubMed Google Scholar, 11.Roberts D.M. Besl L. Oh S.H. Masterson R.V. Schell J. Stacey G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8394-8398Crossref PubMed Scopus (44) Google Scholar, 12.Harding S. Oh S.H. Roberts D.M. EMBO J. 1997; 16: 1137-1144Crossref PubMed Scopus (169) Google Scholar) and might also influence posttranslational ubiquitination of the protein (13.Gregori L. Marriott D. West C.M. Chau V. J. Biol. Chem. 1985; 260: 5232-5235Abstract Full Text PDF PubMed Google Scholar).Previous work shows that the site on calmodulin recognized by the calmodulin N-methyltransferase resides solely on the COOH-terminal lobe (residues 78–148) (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Mutations or chemical modifications that affect the hydrophobic core and conformation of calmodulin disrupt methylation (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar, 14.Han C.H. Roberts D.M. Eur. J. Biochem. 1997; 244: 904-912Crossref PubMed Scopus (2) Google Scholar), and it seems as if the enzyme requires more than a linear sequence of amino acids or the simple surface exposure of lysine 115 for recognition and methylation.An examination of the calmodulin structure shows that the amino-terminal and carboxyl-terminal lobes share remarkable structural similarity and symmetry (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar). In previous work we performed a series of domain duplication and exchange mutagenesis experiments in which EF-hands III and IV were substituted with the symmetry-related EF-hands I and II (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar). These experiments showed that structural features unique to both EF-hands are required for methyltransferase binding and methylation. To define more precisely the regions responsible for calmodulin methyltransferase specificity, we exploited this domain exchange approach further and performed site-directed mutagenesis of various residues in the carboxyl-terminal lobe surrounding the methylation site. The results implicate specific regions in the methylation loop/turn region between EF-hands III and IV as well as residues within the adjacent α-helices in the binding and recognition of the enzyme.DISCUSSIONCalmodulin is trimethylated at lysine 115 with a high degree of specificity by a dedicated calmodulin lysineN-methyltransferase. In the present study, domain exchange and scanning mutagenesis were done to attempt to identify regions of the protein that contribute to this specificity. The results suggest that three structural regions within the carboxyl-terminal lobe appear to be involved: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent α-helices (helix 6 and 7) from EF-hands III and IV.The six-amino acid methylation loop (LGEKLT) is highly conserved among phylogenetically diverse calmodulins, and it is reasonable to suggest that its structure provides features necessary for calmodulin methyltransferase recognition. Structural studies suggest that the loop shows greater flexibility and dynamics compared with the calcium binding loops and helices of the EF-hands (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar, 23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar, 24.Malmendal A. Evenas J. Forsen S. Akke M. J. Mol. Biol. 1999; 293: 883-899Crossref PubMed Scopus (144) Google Scholar). The loop provides a 90° hairpin turn between EF-hands III and IV, which is facilitated by Gly-113 (φ/ψ = 93°/10° (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar)), and its conformation is stabilized by three hydrogen bonds between the backbone amide nitrogens of Gly-113 and Glu-114 and the backbone carbonyl oxygens of Met-109 and Thr-110 in helix 6 of EF-hand III (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar). In addition, Leu-116 is imbedded in the core of the carboxyl-terminal lobe, forming hydrophobic interactions with residues on the hydrophobic faces of the helices from EF-hands III and IV (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar). Glu-114 and Lys-115 are solvent-exposed charged residues with no apparent contacts with other parts of the calmodulin structure (Fig. 5).Calmodulin methylation is exquisitely sensitive to the substitutions of G113S, E114A, and L116T, which essentially abolish the methylation of lysine 115. These defects were observed regardless of calcium concentration, and thus, both the recognition of calcium-bound as well as apo-calmodulin was affected. Based on the structural features of these residues discussed above, some potential roles in methyltransferase recognition can be suggested. The substitution of glutamate 114 with an alanine removes a surface negative charge adjacent to the site of methylation and, as discussed further below, could provide an electrostatic contact for the enzyme. The substitution of a serine for the highly conserved glycine 113 likely alters the conformational flexibility of the loop-turn structure and might prohibit the residues within the loop from adopting an orientation suitable for methyltransferase binding and catalysis. The substitution of L116T, which is one of 14 residues composing the hydrophobic core of the carboxyl-terminal lobe, could alter the packing of the hydrophobic side chains and the interaction of the methylation loop with the hydrophobic core. Interestingly, none of these mutations significantly affects activation of two calmodulin-dependent enzymes, suggesting that their structural effects are subtle, selectively affecting methyltransferase recognition but not other calmodulin functions.The conserved residues of the methylation loop are not in themselves adequate to confer methylation. For example, previous work (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar) showed that the introduction of the methylation loop at a symmetrical position within the amino-terminal lobe did not result in lysine methylation. Furthermore, the replacement of either EF-hand III or IV with the homologous EF-hand I or II also results in the loss of lysine methylation (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar). In the present study, we find that the critical regions are the α-helices adjacent to the methylation site, helix 6 of EF-hand III and helix 7 of EF-hand IV.The substitution of helix 2 (LGTVMRS) for helix 6 (LRHVMTN) resulted in a substantial reduction in the rate of lysine methylation in both the presence and absence of calcium. A comparison of the structure of these related regions shows that they have remarkably similar backbone structures and form nearly identical packing interactions in their respective helical bundles within the amino or carboxyl termini (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar, 23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar,25.Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (612) Google Scholar). Thus, the loss of methylation was thought to be the result of alterations of surface residues that presumably interact with the methyltransferase. However, individual charge-to-neutral substitutions at these positions showed essentially normal methylation. Thus, the conservation of these surface residues is not apparently required for methyltransferase activity; however, the packing interactions of helix 6 with others in the carboxyl-terminal lobe may be important for stabilizing the conformation of the residues that are recognized and bound by the methyltransferase. The substitution of helix 2 apparently perturbs these interactions, a result that was not anticipated based on the similarity of the two structures. The reason for this defect in CaMH6 is not yet clear.In contrast, helix 7 shows a much different influence on the methylation of lysine 115. This helix introduces a high density of electrostatic charge on the carboxyl-terminal lobe (21.Weber P.C. Lukas T.J. Craig T.A. Wilson E. King M.M. Kwiatkowski A.P. Watterson D.M. Proteins. 1989; 6: 70-85Crossref PubMed Scopus (46) Google Scholar) adjacent to the site of methylation (Fig. 5). Mutagenesis of these various charged groups show that the removal of charges at positions 118 and 120 and to a lesser degree from positions 122, 126, and 127 results in a reduction in the rate of methylation. These findings along with the E114A results discussed above suggest that electrostatic interactions may play a role in the binding of the methyltransferase.Interestingly, many of the defects associated with the substitutions within helix 7 apparently are more severe in apoCaM compared with Ca2+-CaM. Additionally, other mutations, such as the L112T substitution within the methylation loop, are only defective in apoCaM. This difference in the recognition of apoCaM and Ca2+-CaM by the calmodulin methyltransferase is supported by several previous findings. For example, the methylation of apoCaM shows different kinetics and considerably greater sensitivity to conditions of increasing ionic strength than Ca2+-CaM (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Conversely, the methylation of Ca2+-CaM, but not apoCaM, is sensitive to peptides and ligands that bind to the hydrophobic cleft (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar, 8.Pech L.L. Nelson D.L. Biochim. Biophys. Acta. 1994; 1199: 183-194Crossref PubMed Scopus (8) Google Scholar, 9.Wright L.S. Bertics P.J. Siegel F.L. J. Biol. Chem. 1996; 271: 12737-12743Abstract Full Text PDF PubMed Scopus (11) Google Scholar). The inability of the cam2 mutant of Paramecium to be methylated normally in vivo (26.Lukas T.J. Friedman M.W. Kung C. Watterson D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7331-7335Crossref PubMed Scopus (30) Google Scholar) was found to be due to an inability to selectively recognize the apo form of calmodulin (14.Han C.H. Roberts D.M. Eur. J. Biochem. 1997; 244: 904-912Crossref PubMed Scopus (2) Google Scholar). Thus, although both Ca2+- and apoCaM are trimethylated by the calmodulin methyltransferase, they interact with the enzyme in a distinct fashion.Based on these previous studies and the present mutagenesis work, we propose a model for the interaction of the methyltransferase with the two forms of calmodulin (Fig. 5). ApoCaM exists predominantly in a closed conformation consisting of the four α-helices of the EF-hand pair packed in an antiparallel (128°-137°) orientation relative to one another (23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar, 25.Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (612) Google Scholar). This results in fewer exposed hydrophobic residues and a high density of surface charge residues (Fig. 5). Based on the charge substitutions, electrostatic interactions between the methyltransferase and the charged residues of helix 7 and the methylation loop of apoCaM may help contribute to binding/orientation of the calmodulin substrate. This supports previous findings that the interaction of apoCaM with the calmodulin methyltransferase (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar) is sensitive to ion concentrations. Interestingly, the interaction of apoCaM with other target proteins shows a similar sensitivity (27.Van Hooff C.O.M. De Graan P.N.E. Oestreicher A.B. Gispen W.H. J. Neurosci. 1988; 8: 1789-1795Crossref PubMed Google Scholar, 28.Baudier J. Deloume J.C. Van Dorsselaer A. Black D. Matthes H.W.D. J. Biol. Chem. 1991; 266: 229-237Abstract Full Text PDF PubMed Google Scholar, 29.Urbauer J.L. Short J.H. Dow L.K. Wand A.J. Biochemistry. 1995; 34: 8099-8109Crossref PubMed Scopus (87) Google Scholar, 30.Tsvetkov P.O. Protasevich I.I. Gilli R. Lafitte D. Lobachov V.M. Haiech J. Briand C. Makarov A.A. J. Biol. Chem. 1999; 274: 18161-18164Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).The binding of calcium induces a conformational change in the lobe, resulting in a shift of the EF-hand interhelical angles to an almost perpendicular state (86°-101°). Although the relative conformation of helix 6-methylation loop helix 7 (residues 106–126) undergoes a small change between apoCaM and Ca2+-CaM (root mean square deviation is 2.6 Å), the major change between the two structures is the surface exposure of a pronounced hydrophobic pocket adjacent to the site of methylation (Fig. 5). This surface might provide additional interactions with the methyltransferase. This could explain why reagents such as drugs and peptides, which interact selectively with the hydrophobic cleft, block the binding of the methyltransferase to Ca2+-CaM but not to apoCaM (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Furthermore, this could also help explain why several of the charge-to-alanine mutations have less severe effects on calmodulin methylation in the presence of calcium. Calmodulin is a highly conserved calcium sensor protein that modulates the activities of multiple enzymes. Calmodulin is a monomer consisting of two structurally similar globular calcium binding lobes (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar) connected by a flexible linker region (2.Persechini A. Kretsinger R.H. J. Biol. Chem. 1988; 263: 12175-12178Abstract Full Text PDF PubMed Google Scholar, 3.Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (887) Google Scholar). Each lobe consists of two helix-loop-helix EF-hand calcium binding sites, with EF-hand domains I and II constituting the amino-terminal lobe and EF-hands III and IV constituting the carboxyl-terminal lobe. Many naturally occurring calmodulins are posttranslationally trimethylated on a single lysine residue at position 115 (reviewed in Ref. 4.Siegel F.L. Vincent P.L. Neal T.L. Wright L.S. Heth A.A. Rowe P.M. Paik W.K. Kim S. Protein Methylation. CRC Press, Inc., Boca Raton, FL1990: 33-58Google Scholar). Lysine 115 is a solvent-exposed residue that is found on a highly conserved six-amino acid loop-turn region (LGEKLT) located between helix 6 of EF-hand III and helix 7 of EF-hand IV (Fig. 1). Trimethylation of calmodulin at lysine 115 is catalyzed by an N-methyltransferase that utilizes S-adenosylmethionine as a co-substrate (Refs. 5.Rowe P.M. Wright L.S. Siegel F.L. J. Biol. Chem. 1986; 261: 7060-7069Abstract Full Text PDF PubMed Google Scholar, 6.Morino H. Kawamoto T. Miyake M. Kakimoto Y. J. Neurochem. 1987; 48: 1201-1208Crossref PubMed Scopus (18) Google Scholar, 7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar, 8.Pech L.L. Nelson D.L. Biochim. Biophys. Acta. 1994; 1199: 183-194Crossref PubMed Scopus (8) Google Scholar, 9.Wright L.S. Bertics P.J. Siegel F.L. J. Biol. Chem. 1996; 271: 12737-12743Abstract Full Text PDF PubMed Scopus (11) Google Scholar, reviewed in Ref. 4.Siegel F.L. Vincent P.L. Neal T.L. Wright L.S. Heth A.A. Rowe P.M. Paik W.K. Kim S. Protein Methylation. CRC Press, Inc., Boca Raton, FL1990: 33-58Google Scholar). This enzyme appears to have the dedicated function of trimethylating lysine 115 in calmodulin from a wide variety of species. From a functional perspective, calmodulin methylation selectively affects the regulation of certain enzymes such as NAD kinase (10.Roberts D.M. Rowe P.M. Siegel F.L. Lukas T.J. Watterson D.M. J. Biol. Chem. 1986; 261: 1491-1494Abstract Full Text PDF PubMed Google Scholar, 11.Roberts D.M. Besl L. Oh S.H. Masterson R.V. Schell J. Stacey G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8394-8398Crossref PubMed Scopus (44) Google Scholar, 12.Harding S. Oh S.H. Roberts D.M. EMBO J. 1997; 16: 1137-1144Crossref PubMed Scopus (169) Google Scholar) and might also influence posttranslational ubiquitination of the protein (13.Gregori L. Marriott D. West C.M. Chau V. J. Biol. Chem. 1985; 260: 5232-5235Abstract Full Text PDF PubMed Google Scholar). Previous work shows that the site on calmodulin recognized by the calmodulin N-methyltransferase resides solely on the COOH-terminal lobe (residues 78–148) (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Mutations or chemical modifications that affect the hydrophobic core and conformation of calmodulin disrupt methylation (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar, 14.Han C.H. Roberts D.M. Eur. J. Biochem. 1997; 244: 904-912Crossref PubMed Scopus (2) Google Scholar), and it seems as if the enzyme requires more than a linear sequence of amino acids or the simple surface exposure of lysine 115 for recognition and methylation. An examination of the calmodulin structure shows that the amino-terminal and carboxyl-terminal lobes share remarkable structural similarity and symmetry (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar). In previous work we performed a series of domain duplication and exchange mutagenesis experiments in which EF-hands III and IV were substituted with the symmetry-related EF-hands I and II (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar). These experiments showed that structural features unique to both EF-hands are required for methyltransferase binding and methylation. To define more precisely the regions responsible for calmodulin methyltransferase specificity, we exploited this domain exchange approach further and performed site-directed mutagenesis of various residues in the carboxyl-terminal lobe surrounding the methylation site. The results implicate specific regions in the methylation loop/turn region between EF-hands III and IV as well as residues within the adjacent α-helices in the binding and recognition of the enzyme. DISCUSSIONCalmodulin is trimethylated at lysine 115 with a high degree of specificity by a dedicated calmodulin lysineN-methyltransferase. In the present study, domain exchange and scanning mutagenesis were done to attempt to identify regions of the protein that contribute to this specificity. The results suggest that three structural regions within the carboxyl-terminal lobe appear to be involved: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent α-helices (helix 6 and 7) from EF-hands III and IV.The six-amino acid methylation loop (LGEKLT) is highly conserved among phylogenetically diverse calmodulins, and it is reasonable to suggest that its structure provides features necessary for calmodulin methyltransferase recognition. Structural studies suggest that the loop shows greater flexibility and dynamics compared with the calcium binding loops and helices of the EF-hands (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar, 23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar, 24.Malmendal A. Evenas J. Forsen S. Akke M. J. Mol. Biol. 1999; 293: 883-899Crossref PubMed Scopus (144) Google Scholar). The loop provides a 90° hairpin turn between EF-hands III and IV, which is facilitated by Gly-113 (φ/ψ = 93°/10° (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar)), and its conformation is stabilized by three hydrogen bonds between the backbone amide nitrogens of Gly-113 and Glu-114 and the backbone carbonyl oxygens of Met-109 and Thr-110 in helix 6 of EF-hand III (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar). In addition, Leu-116 is imbedded in the core of the carboxyl-terminal lobe, forming hydrophobic interactions with residues on the hydrophobic faces of the helices from EF-hands III and IV (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar). Glu-114 and Lys-115 are solvent-exposed charged residues with no apparent contacts with other parts of the calmodulin structure (Fig. 5).Calmodulin methylation is exquisitely sensitive to the substitutions of G113S, E114A, and L116T, which essentially abolish the methylation of lysine 115. These defects were observed regardless of calcium concentration, and thus, both the recognition of calcium-bound as well as apo-calmodulin was affected. Based on the structural features of these residues discussed above, some potential roles in methyltransferase recognition can be suggested. The substitution of glutamate 114 with an alanine removes a surface negative charge adjacent to the site of methylation and, as discussed further below, could provide an electrostatic contact for the enzyme. The substitution of a serine for the highly conserved glycine 113 likely alters the conformational flexibility of the loop-turn structure and might prohibit the residues within the loop from adopting an orientation suitable for methyltransferase binding and catalysis. The substitution of L116T, which is one of 14 residues composing the hydrophobic core of the carboxyl-terminal lobe, could alter the packing of the hydrophobic side chains and the interaction of the methylation loop with the hydrophobic core. Interestingly, none of these mutations significantly affects activation of two calmodulin-dependent enzymes, suggesting that their structural effects are subtle, selectively affecting methyltransferase recognition but not other calmodulin functions.The conserved residues of the methylation loop are not in themselves adequate to confer methylation. For example, previous work (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar) showed that the introduction of the methylation loop at a symmetrical position within the amino-terminal lobe did not result in lysine methylation. Furthermore, the replacement of either EF-hand III or IV with the homologous EF-hand I or II also results in the loss of lysine methylation (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar). In the present study, we find that the critical regions are the α-helices adjacent to the methylation site, helix 6 of EF-hand III and helix 7 of EF-hand IV.The substitution of helix 2 (LGTVMRS) for helix 6 (LRHVMTN) resulted in a substantial reduction in the rate of lysine methylation in both the presence and absence of calcium. A comparison of the structure of these related regions shows that they have remarkably similar backbone structures and form nearly identical packing interactions in their respective helical bundles within the amino or carboxyl termini (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar, 23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar,25.Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (612) Google Scholar). Thus, the loss of methylation was thought to be the result of alterations of surface residues that presumably interact with the methyltransferase. However, individual charge-to-neutral substitutions at these positions showed essentially normal methylation. Thus, the conservation of these surface residues is not apparently required for methyltransferase activity; however, the packing interactions of helix 6 with others in the carboxyl-terminal lobe may be important for stabilizing the conformation of the residues that are recognized and bound by the methyltransferase. The substitution of helix 2 apparently perturbs these interactions, a result that was not anticipated based on the similarity of the two structures. The reason for this defect in CaMH6 is not yet clear.In contrast, helix 7 shows a much different influence on the methylation of lysine 115. This helix introduces a high density of electrostatic charge on the carboxyl-terminal lobe (21.Weber P.C. Lukas T.J. Craig T.A. Wilson E. King M.M. Kwiatkowski A.P. Watterson D.M. Proteins. 1989; 6: 70-85Crossref PubMed Scopus (46) Google Scholar) adjacent to the site of methylation (Fig. 5). Mutagenesis of these various charged groups show that the removal of charges at positions 118 and 120 and to a lesser degree from positions 122, 126, and 127 results in a reduction in the rate of methylation. These findings along with the E114A results discussed above suggest that electrostatic interactions may play a role in the binding of the methyltransferase.Interestingly, many of the defects associated with the substitutions within helix 7 apparently are more severe in apoCaM compared with Ca2+-CaM. Additionally, other mutations, such as the L112T substitution within the methylation loop, are only defective in apoCaM. This difference in the recognition of apoCaM and Ca2+-CaM by the calmodulin methyltransferase is supported by several previous findings. For example, the methylation of apoCaM shows different kinetics and considerably greater sensitivity to conditions of increasing ionic strength than Ca2+-CaM (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Conversely, the methylation of Ca2+-CaM, but not apoCaM, is sensitive to peptides and ligands that bind to the hydrophobic cleft (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar, 8.Pech L.L. Nelson D.L. Biochim. Biophys. Acta. 1994; 1199: 183-194Crossref PubMed Scopus (8) Google Scholar, 9.Wright L.S. Bertics P.J. Siegel F.L. J. Biol. Chem. 1996; 271: 12737-12743Abstract Full Text PDF PubMed Scopus (11) Google Scholar). The inability of the cam2 mutant of Paramecium to be methylated normally in vivo (26.Lukas T.J. Friedman M.W. Kung C. Watterson D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7331-7335Crossref PubMed Scopus (30) Google Scholar) was found to be due to an inability to selectively recognize the apo form of calmodulin (14.Han C.H. Roberts D.M. Eur. J. Biochem. 1997; 244: 904-912Crossref PubMed Scopus (2) Google Scholar). Thus, although both Ca2+- and apoCaM are trimethylated by the calmodulin methyltransferase, they interact with the enzyme in a distinct fashion.Based on these previous studies and the present mutagenesis work, we propose a model for the interaction of the methyltransferase with the two forms of calmodulin (Fig. 5). ApoCaM exists predominantly in a closed conformation consisting of the four α-helices of the EF-hand pair packed in an antiparallel (128°-137°) orientation relative to one another (23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar, 25.Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (612) Google Scholar). This results in fewer exposed hydrophobic residues and a high density of surface charge residues (Fig. 5). Based on the charge substitutions, electrostatic interactions between the methyltransferase and the charged residues of helix 7 and the methylation loop of apoCaM may help contribute to binding/orientation of the calmodulin substrate. This supports previous findings that the interaction of apoCaM with the calmodulin methyltransferase (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar) is sensitive to ion concentrations. Interestingly, the interaction of apoCaM with other target proteins shows a similar sensitivity (27.Van Hooff C.O.M. De Graan P.N.E. Oestreicher A.B. Gispen W.H. J. Neurosci. 1988; 8: 1789-1795Crossref PubMed Google Scholar, 28.Baudier J. Deloume J.C. Van Dorsselaer A. Black D. Matthes H.W.D. J. Biol. Chem. 1991; 266: 229-237Abstract Full Text PDF PubMed Google Scholar, 29.Urbauer J.L. Short J.H. Dow L.K. Wand A.J. Biochemistry. 1995; 34: 8099-8109Crossref PubMed Scopus (87) Google Scholar, 30.Tsvetkov P.O. Protasevich I.I. Gilli R. Lafitte D. Lobachov V.M. Haiech J. Briand C. Makarov A.A. J. Biol. Chem. 1999; 274: 18161-18164Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).The binding of calcium induces a conformational change in the lobe, resulting in a shift of the EF-hand interhelical angles to an almost perpendicular state (86°-101°). Although the relative conformation of helix 6-methylation loop helix 7 (residues 106–126) undergoes a small change between apoCaM and Ca2+-CaM (root mean square deviation is 2.6 Å), the major change between the two structures is the surface exposure of a pronounced hydrophobic pocket adjacent to the site of methylation (Fig. 5). This surface might provide additional interactions with the methyltransferase. This could explain why reagents such as drugs and peptides, which interact selectively with the hydrophobic cleft, block the binding of the methyltransferase to Ca2+-CaM but not to apoCaM (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Furthermore, this could also help explain why several of the charge-to-alanine mutations have less severe effects on calmodulin methylation in the presence of calcium. Calmodulin is trimethylated at lysine 115 with a high degree of specificity by a dedicated calmodulin lysineN-methyltransferase. In the present study, domain exchange and scanning mutagenesis were done to attempt to identify regions of the protein that contribute to this specificity. The results suggest that three structural regions within the carboxyl-terminal lobe appear to be involved: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent α-helices (helix 6 and 7) from EF-hands III and IV. The six-amino acid methylation loop (LGEKLT) is highly conserved among phylogenetically diverse calmodulins, and it is reasonable to suggest that its structure provides features necessary for calmodulin methyltransferase recognition. Structural studies suggest that the loop shows greater flexibility and dynamics compared with the calcium binding loops and helices of the EF-hands (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar, 23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar, 24.Malmendal A. Evenas J. Forsen S. Akke M. J. Mol. Biol. 1999; 293: 883-899Crossref PubMed Scopus (144) Google Scholar). The loop provides a 90° hairpin turn between EF-hands III and IV, which is facilitated by Gly-113 (φ/ψ = 93°/10° (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar)), and its conformation is stabilized by three hydrogen bonds between the backbone amide nitrogens of Gly-113 and Glu-114 and the backbone carbonyl oxygens of Met-109 and Thr-110 in helix 6 of EF-hand III (22.Chattopadhyaya R. Meador W.E. Means A.R. Quiocho F.A. J. Mol. Biol. 1992; 228: 1177-1192Crossref PubMed Scopus (614) Google Scholar). In addition, Leu-116 is imbedded in the core of the carboxyl-terminal lobe, forming hydrophobic interactions with residues on the hydrophobic faces of the helices from EF-hands III and IV (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar). Glu-114 and Lys-115 are solvent-exposed charged residues with no apparent contacts with other parts of the calmodulin structure (Fig. 5). Calmodulin methylation is exquisitely sensitive to the substitutions of G113S, E114A, and L116T, which essentially abolish the methylation of lysine 115. These defects were observed regardless of calcium concentration, and thus, both the recognition of calcium-bound as well as apo-calmodulin was affected. Based on the structural features of these residues discussed above, some potential roles in methyltransferase recognition can be suggested. The substitution of glutamate 114 with an alanine removes a surface negative charge adjacent to the site of methylation and, as discussed further below, could provide an electrostatic contact for the enzyme. The substitution of a serine for the highly conserved glycine 113 likely alters the conformational flexibility of the loop-turn structure and might prohibit the residues within the loop from adopting an orientation suitable for methyltransferase binding and catalysis. The substitution of L116T, which is one of 14 residues composing the hydrophobic core of the carboxyl-terminal lobe, could alter the packing of the hydrophobic side chains and the interaction of the methylation loop with the hydrophobic core. Interestingly, none of these mutations significantly affects activation of two calmodulin-dependent enzymes, suggesting that their structural effects are subtle, selectively affecting methyltransferase recognition but not other calmodulin functions. The conserved residues of the methylation loop are not in themselves adequate to confer methylation. For example, previous work (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar) showed that the introduction of the methylation loop at a symmetrical position within the amino-terminal lobe did not result in lysine methylation. Furthermore, the replacement of either EF-hand III or IV with the homologous EF-hand I or II also results in the loss of lysine methylation (15.Cobb J.A. Han C.H. Wills D.M. Roberts D.M. Biochem. J. 1999; 340: 417-424PubMed Google Scholar). In the present study, we find that the critical regions are the α-helices adjacent to the methylation site, helix 6 of EF-hand III and helix 7 of EF-hand IV. The substitution of helix 2 (LGTVMRS) for helix 6 (LRHVMTN) resulted in a substantial reduction in the rate of lysine methylation in both the presence and absence of calcium. A comparison of the structure of these related regions shows that they have remarkably similar backbone structures and form nearly identical packing interactions in their respective helical bundles within the amino or carboxyl termini (1.Babu Y.S. Bugg C.E. Cook W.J. J. Mol. Biol. 1988; 204: 191-204Crossref PubMed Scopus (967) Google Scholar, 23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar,25.Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (612) Google Scholar). Thus, the loss of methylation was thought to be the result of alterations of surface residues that presumably interact with the methyltransferase. However, individual charge-to-neutral substitutions at these positions showed essentially normal methylation. Thus, the conservation of these surface residues is not apparently required for methyltransferase activity; however, the packing interactions of helix 6 with others in the carboxyl-terminal lobe may be important for stabilizing the conformation of the residues that are recognized and bound by the methyltransferase. The substitution of helix 2 apparently perturbs these interactions, a result that was not anticipated based on the similarity of the two structures. The reason for this defect in CaMH6 is not yet clear. In contrast, helix 7 shows a much different influence on the methylation of lysine 115. This helix introduces a high density of electrostatic charge on the carboxyl-terminal lobe (21.Weber P.C. Lukas T.J. Craig T.A. Wilson E. King M.M. Kwiatkowski A.P. Watterson D.M. Proteins. 1989; 6: 70-85Crossref PubMed Scopus (46) Google Scholar) adjacent to the site of methylation (Fig. 5). Mutagenesis of these various charged groups show that the removal of charges at positions 118 and 120 and to a lesser degree from positions 122, 126, and 127 results in a reduction in the rate of methylation. These findings along with the E114A results discussed above suggest that electrostatic interactions may play a role in the binding of the methyltransferase. Interestingly, many of the defects associated with the substitutions within helix 7 apparently are more severe in apoCaM compared with Ca2+-CaM. Additionally, other mutations, such as the L112T substitution within the methylation loop, are only defective in apoCaM. This difference in the recognition of apoCaM and Ca2+-CaM by the calmodulin methyltransferase is supported by several previous findings. For example, the methylation of apoCaM shows different kinetics and considerably greater sensitivity to conditions of increasing ionic strength than Ca2+-CaM (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Conversely, the methylation of Ca2+-CaM, but not apoCaM, is sensitive to peptides and ligands that bind to the hydrophobic cleft (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar, 8.Pech L.L. Nelson D.L. Biochim. Biophys. Acta. 1994; 1199: 183-194Crossref PubMed Scopus (8) Google Scholar, 9.Wright L.S. Bertics P.J. Siegel F.L. J. Biol. Chem. 1996; 271: 12737-12743Abstract Full Text PDF PubMed Scopus (11) Google Scholar). The inability of the cam2 mutant of Paramecium to be methylated normally in vivo (26.Lukas T.J. Friedman M.W. Kung C. Watterson D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7331-7335Crossref PubMed Scopus (30) Google Scholar) was found to be due to an inability to selectively recognize the apo form of calmodulin (14.Han C.H. Roberts D.M. Eur. J. Biochem. 1997; 244: 904-912Crossref PubMed Scopus (2) Google Scholar). Thus, although both Ca2+- and apoCaM are trimethylated by the calmodulin methyltransferase, they interact with the enzyme in a distinct fashion. Based on these previous studies and the present mutagenesis work, we propose a model for the interaction of the methyltransferase with the two forms of calmodulin (Fig. 5). ApoCaM exists predominantly in a closed conformation consisting of the four α-helices of the EF-hand pair packed in an antiparallel (128°-137°) orientation relative to one another (23.Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (643) Google Scholar, 25.Kuboniwa H. Tjandra N. Grzesiek S. Ren H. Klee C.B. Bax A. Nat. Struct. Biol. 1995; 2: 768-776Crossref PubMed Scopus (612) Google Scholar). This results in fewer exposed hydrophobic residues and a high density of surface charge residues (Fig. 5). Based on the charge substitutions, electrostatic interactions between the methyltransferase and the charged residues of helix 7 and the methylation loop of apoCaM may help contribute to binding/orientation of the calmodulin substrate. This supports previous findings that the interaction of apoCaM with the calmodulin methyltransferase (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar) is sensitive to ion concentrations. Interestingly, the interaction of apoCaM with other target proteins shows a similar sensitivity (27.Van Hooff C.O.M. De Graan P.N.E. Oestreicher A.B. Gispen W.H. J. Neurosci. 1988; 8: 1789-1795Crossref PubMed Google Scholar, 28.Baudier J. Deloume J.C. Van Dorsselaer A. Black D. Matthes H.W.D. J. Biol. Chem. 1991; 266: 229-237Abstract Full Text PDF PubMed Google Scholar, 29.Urbauer J.L. Short J.H. Dow L.K. Wand A.J. Biochemistry. 1995; 34: 8099-8109Crossref PubMed Scopus (87) Google Scholar, 30.Tsvetkov P.O. Protasevich I.I. Gilli R. Lafitte D. Lobachov V.M. Haiech J. Briand C. Makarov A.A. J. Biol. Chem. 1999; 274: 18161-18164Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The binding of calcium induces a conformational change in the lobe, resulting in a shift of the EF-hand interhelical angles to an almost perpendicular state (86°-101°). Although the relative conformation of helix 6-methylation loop helix 7 (residues 106–126) undergoes a small change between apoCaM and Ca2+-CaM (root mean square deviation is 2.6 Å), the major change between the two structures is the surface exposure of a pronounced hydrophobic pocket adjacent to the site of methylation (Fig. 5). This surface might provide additional interactions with the methyltransferase. This could explain why reagents such as drugs and peptides, which interact selectively with the hydrophobic cleft, block the binding of the methyltransferase to Ca2+-CaM but not to apoCaM (7.Han C.H. Richardson J. Oh S.H. Roberts D.M. Biochemistry. 1993; 32: 13974-13980Crossref PubMed Scopus (12) Google Scholar). Furthermore, this could also help explain why several of the charge-to-alanine mutations have less severe effects on calmodulin methylation in the presence of calcium.