Roles of Active Site Residues and the HUH Motif of the F Plasmid TraI Relaxase

Abstract

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA. Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA. Bacterial conjugation contributes to diversification of prokaryotic genomes through horizontal transfer of conjugative plasmids between bacteria. During conjugation, a single strand of the plasmid is cleaved in the donor cell and transferred to the recipient bacterium using plasmid-encoded secretion machinery (1Zechner E.L. de la Cruz F. Eisenbrandt R. Grahn A.M. Koraimann G. Lanka E. Muth G. Pansegrau W. Thomas C.M. Wilkins B.M. Zatyka M. Thomas C.M. The Horizontal Gene Pool. Harwood Academic Publishers, Amsterdam, The Netherlands2000: 87-174Google Scholar). Conjugation may play important roles in evolution of pathogenic Escherichia coli (2Wirth T. Falush D. Lan R. Colles F. Mensa P. Wieler L.H. Karch H. Reeves P.R. Maiden M.C. Ochman H. Achtman M. Mol. Microbiol. 2006; 60: 1136-1151Crossref PubMed Scopus (1466) Google Scholar) and the formation of biofilms (3Reisner A. Holler B.M. Molin S. Zechner E.L. J. Bacteriol. 2006; 188: 3582-3588Crossref PubMed Scopus (110) Google Scholar). Despite its importance in genetic diversification and the acquisition of new traits, however, the mechanism of bacterial conjugation is still a relatively poorly understood process. Initiation of conjugation requires strand- and site-specific cleavage of the plasmid within the origin of transfer (oriT) at a site termed nic. The relaxase or mobilization (Mob) protein encoded by the plasmid carries out the metal-dependent cleavage reaction. TraI is the relaxase for F plasmid (4Reygers U. Wessel R. Muller H. Hoffmann-Berling H. EMBO J. 1991; 10: 2689-2694Crossref PubMed Scopus (61) Google Scholar, 5Matson S.W. Morton B.S. J. Biol. Chem. 1991; 266: 16232-16237Abstract Full Text PDF PubMed Google Scholar). The cleavage reaction is facilitated by accessory proteins that recruit TraI to oriT, perhaps by directly interacting with the relaxase or creating a favorable DNA conformation for the relaxase to bind (6Fekete R.A. Frost L.S. J. Bacteriol. 2000; 182: 4022-4027Crossref PubMed Scopus (33) Google Scholar, 7Nelson W.C. Howard M.T. Sherman J.A. Matson S.W. J. Biol. Chem. 1995; 270: 28374-28380Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 8Howard M.T. Nelson W.C. Matson S.W. J. Biol. Chem. 1995; 270: 28381-28386Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The cleavage reaction results in a long-lived physical link between the plasmid and the relaxase, through a phosphotyrosyl bond bridging the catalytic tyrosine residue and the DNA backbone (9Byrd D.R. Matson S.W. Mol. Microbiol. 1997; 25: 1011-1022Crossref PubMed Scopus (97) Google Scholar, 10Sherman J.A. Matson S.W. J. Biol. Chem. 1994; 269: 26220-26226Abstract Full Text PDF PubMed Google Scholar). After transfer of the plasmid to the recipient, possibly as a protein-DNA conjugate (11Draper O. Cesar C.E. Machon C. de la Cruz F. Llosa M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16385-16390Crossref PubMed Scopus (88) Google Scholar), the cut plasmid is recircularized, most likely by the relaxase, and second strand synthesis occurs. The process produces a donor and recipient that are both competent for further transfer of the plasmid. F TraI is a 192-kDa bifunctional protein essential for bacterial conjugation (4Reygers U. Wessel R. Muller H. Hoffmann-Berling H. EMBO J. 1991; 10: 2689-2694Crossref PubMed Scopus (61) Google Scholar). The N-terminal 330 amino acids contain the specific single-stranded DNA (ssDNA) 5The abbreviations used are: ssDNA, single-stranded DNA; ITC, isothermal titration calorimetry. recognition and cleavage activities of the protein, whereas the helicase activity is contained in the C-terminal region (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google Scholar, 13Byrd D.R. Sampson J.K. Ragonese H.M. Matson S.W. J. Biol. Chem. 2002; 277: 42645-42653Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The relaxase and helicase activities must be linked for efficient plasmid transfer (13Byrd D.R. Sampson J.K. Ragonese H.M. Matson S.W. J. Biol. Chem. 2002; 277: 42645-42653Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Most relaxases have a His-hydrophobe-His (HUH) motif that, when first identified, was proposed to be a metal-binding site (14Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (504) Google Scholar). This HUH motif is conserved in plasmid and viral initiation factors (15Koonin E.V. Ilyina T.V. Biosystems. 1993; 30: 241-268Crossref PubMed Scopus (152) Google Scholar). F TraI and relaxases from related plasmids, including TraI from R100 and R1, and R388 TrwC, are further defined by an YY-X5-6-YY motif located in the N-terminal portion of the protein. This motif includes the catalytic Tyr and other residues important for relaxase function (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google Scholar, 16Boer R. Russi S. Guasch A. Lucas M. Blanco A.G. Perez-Luque R. Coll M. de la Cruz F. J. Mol. Biol. 2006; 358: 857-869Crossref PubMed Scopus (62) Google Scholar). Recent structures of members of this family of proteins have shown that the HUH motif participates in metal coordination (16Boer R. Russi S. Guasch A. Lucas M. Blanco A.G. Perez-Luque R. Coll M. de la Cruz F. J. Mol. Biol. 2006; 358: 857-869Crossref PubMed Scopus (62) Google Scholar, 17Hickman A.B. Ronning D.R. Kotin R.M. Dyda F. Mol. Cell. 2002; 10: 327-337Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18Larkin C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Monzingo A.F. Ozburn A. Xia S. Meyer R.J. Robertus J.D. J. Mol. Biol. PubMed Scopus Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar, L.M. Ragonese H. Matson S.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The His residues in the HUH motif have shown to be for efficient conjugation facilitated by R388 A. Lucas M. G. M. Perez-Luque R. de la Cruz F. Coll M. Biol. 2003; 10: PubMed Scopus Google Scholar). however, the function of the HUH motif are The metal for relaxases is the proteins are to a of metal to the in vitro ssDNA cleavage reaction (16Boer R. Russi S. Guasch A. Lucas M. Blanco A.G. Perez-Luque R. Coll M. de la Cruz F. J. Mol. Biol. 2006; 358: 857-869Crossref PubMed Scopus (62) Google Scholar, 18Larkin C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar). and as as that are in E. including that the TraI relaxase is with and at and C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar). are to the of these in E. coli C.E. T.V. PubMed Scopus Google Scholar). have in the S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and ssDNA C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) structures to a bound as have and L.M. Ragonese H. Matson S.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) in structure of a F TraI relaxase In the relaxase a bound A.F. Ozburn A. Xia S. Meyer R.J. Robertus J.D. J. Mol. Biol. PubMed Scopus Google Scholar), and a cluster a in one structure of an bacterial M.J. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a N-terminal TraI that the ssDNA binding and cleavage of the protein (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google Scholar). further the of the cleavage reaction of TraI and the role of the HUH motif in that we have that have substitutions for conserved residues in the including the HUH motif and residues. have the of the to bind ssDNA, and to single-stranded DNA and to bacterial conjugation in In TraI the substitutions with for and reduced cleavage of ssDNA and transfer with reduced efficiencies. The results suggest that the metal binding cluster and the residues that with a and to the of positioning a divalent cation and scissile phosphate in the of sequence-specific ssDNA and for proteins from the plasmid (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google Scholar) using the plasmids by of are and and as (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google Scholar). for TraI by a with and to a the N-terminal portion of the protein with the substitution of to a from the that the protein (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google Scholar). The for by and the of by DNA of and proteins by in of a single-stranded protein binding. The was by DNA and as J.C. Schildbach J.F. PubMed Scopus Google Scholar). The was to in binding or as was in an to a single binding site using and the is the is the is the of ssDNA, is the of protein, and is the DNA cleavage activity of and was using a DNA was with or protein in cleavage with or for at by to to a and results using a of cleaved and using Scholar) and of cleaved was was for each reaction of titration to affinity for was using a was with of in the protein and metal the the of was to in the and with at at was out by to a single site as defined in the conjugation was as R.J. G. T. M. Zechner E.L. J. Bacteriol. 2006; 188: PubMed Scopus (29) Google Scholar), with or was the (14Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (504) Google Scholar), that the HUH motif the metal in viral initiation proteins and relaxases A.B. Ronning D.R. Kotin R.M. Dyda F. Mol. Cell. 2002; 10: 327-337Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18Larkin C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Monzingo A.F. Ozburn A. Xia S. Meyer R.J. Robertus J.D. J. Mol. Biol. PubMed Scopus Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar, L.M. Ragonese H. Matson S.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. Lucas M. G. M. Perez-Luque R. de la Cruz F. Coll M. Biol. 2003; 10: PubMed Scopus Google Scholar). in the F and the TraI metal-binding site C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The HUH motif includes and binds ssDNA, the bridging and a of the scissile phosphate the bound metal the ssDNA and the scissile phosphate for efficient by the Relaxases have a conserved acidic residue near the metal-binding In with both and the roles of the His and the residues with with single amino substitutions and of and proteins for by isothermal titration of the proteins for an ssDNA by in of a binding for as by isothermal titration and of the of binding was to and of the of binding was to in a new of for and of the of and of the of and of the of and of the of in a new of His binding of the the importance of the a of for C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). for results in binding titration of and a in ssDNA Asp and for as these residues are in structures 2002; PubMed Scopus (113) Google Scholar). The near ssDNA In an affinity for that is to ITC, whereas the affinity for ssDNA is reduced by to the for binding to and affinity for ssDNA bind whereas the a reduced affinity for to of and for ssDNA, however, are near binding for ssDNA, with a affinity the of the substitutions for the His the of the proteins to a F tested cleavage activities at and protein in the of or activity with both at these we the activity of the protein using a single metal. The affinity of for is of its affinity for in the of ssDNA C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google we metal in the a with significantly reduced affinity for metal still activity in the of cleavage using a in a was for each of the at the cleaved ssDNA was of the cleavage activities of site is for of protein and at is cleavage at is for of protein and at is cleavage at in a new and cleavage activity with despite reduced for significantly reduced activities with reduced activity with and a with The reduced activity may its reduced affinity for ssDNA, or may from a of the metal by the Asp this second is this the we to have its cleavage activity The cleaved DNA with and with and cleavage The cleavage activity of the with with the reduced activities of and the and the different of and that the His contribute asymmetrically to metal binding and with important the to function. DNA in the of metal binding by in vitro cleavage that the proteins metal The interactions between ssDNA phosphate and the bound metal in the structure C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) suggest that the metal the affinity of for ssDNA. metal binding in the of ssDNA we ssDNA affinity in the and of The affinity of for of a the of the affinity of for ssDNA J.C. Anderson B.J. Schildbach J.F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In we used the affinity reduced ssDNA cleavage activity and J.C. Schildbach J.F. PubMed Scopus Google Scholar), to the of cleavage of the the for the in to in the we affinity for ssDNA is near the of affinity for is in or a of the to of bound affinity of for ssDNA when in with of the and used J.C. Schildbach J.F. PubMed Scopus Google Scholar). The used the affinity to the bound metal. and for an of metal DNA affinity in metal a of a of in the of a of without and a of with we an of ssDNA binding by these despite the proteins affinity for by the affinity of the for ssDNA in the and of The a in the of reduced affinity in the of The affinity of for to metal binding to be by the of bound ssDNA, at at the The affinity of for ssDNA is reduced in the of likely to interactions between and ssDNA that must be to binding. The of ssDNA affinity is for in and by the favorable interactions between the metal bound to the HUH site and the of the scissile by His the of TraI to F plasmid in TraI and used in transfer to an plasmid that with with TraI transfer a relatively to with transfer of site from in a new The transfer results from the the importance of and residues to and The and are and in transfer despite or cleavage in the with transfer from The despite its metal binding ITC, is in with from The reduced transfer of to is with the from the substitution in A. Lucas M. G. M. Perez-Luque R. de la Cruz F. Coll M. Biol. 2003; 10: PubMed Scopus Google Scholar). Whereas in vitro metal binding and ssDNA cleavage the transfer facilitated by that its affinity for metal and the in vivo metal is for the protein to bind metal and the plasmid. from the may suggest that is the metal by TraI in and poorly a in transfer in vitro ssDNA cleavage activity with reduced activity with The between cleavage results and transfer may that is the metal in however, the significantly of metal to for in in vitro Whereas TraI substitutions for for or the transfer for and a at the position transfer A. Lucas M. G. M. Perez-Luque R. de la Cruz F. Coll M. Biol. 2003; 10: PubMed Scopus Google Scholar). is conserved bacterial is with an acidic residue in the viral initiation factors (14Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (504) Google Scholar). the residue is to substitution at the position in in vitro cleavage M. R.M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The reduced transfer with be to reduced metal binding. The of His at this position in whereas and Asp are in viral initiation however, a that the catalytic Tyr of reduced in vitro cleavage (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google however, plasmid transfer with an reduced from TraI and that facilitated by results with A. Lucas M. G. M. Perez-Luque R. de la Cruz F. Coll M. Biol. 2003; 10: PubMed Scopus Google Scholar, G. P. A. Llosa M. de la Cruz F. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). from extensive of that is in cleavage activity G. P. A. Llosa M. de la Cruz F. J. Mol. Biol. 2000; PubMed Scopus Google Scholar), and result the same is for F This result is to with the to the scissile phosphate for cleavage. however, in the region that includes as by the factors in the and in the C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar), may amino in the region to the catalytic when is with both and binds with affinity its affinity for ssDNA C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, S. Larkin C. Schildbach J.F. Structure (Camb.). 2003; Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In binds with and ssDNA with affinity to Substitutions for the cleavage activity of the protein. The activity with activity with The a significantly activity with and activity with The activity of the to the may result from the ssDNA affinity of these have for different for ssDNA, however, is and plasmid transfer with a in Substitutions for the residue A. Lucas M. G. M. Perez-Luque R. de la Cruz F. Coll M. Biol. 2003; 10: PubMed Scopus Google Scholar). The of cleavage of ssDNA with and a in cleavage with M. R.M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). suggest that the acidic residue a His to the and modulates the of the His the of the scissile TraI may be required to that the is an to with the metal. this the role of however, we the substitution to have a metal binding and function. The is of as a bond from a and a bond donor to the would the conformation that would these In to results from other the is to TraI a in transfer A.F. Ozburn A. Xia S. Meyer R.J. Robertus J.D. J. Mol. Biol. PubMed Scopus Google Scholar). In the structures of TraI C. Datta S. Harley M.J. Anderson B.J. Ebie A. Hargreaves V. Schildbach J.F. Structure (Camb.). 2005; 13: 1533-1544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and (16Boer R. Russi S. Guasch A. Lucas M. Blanco A.G. Perez-Luque R. Coll M. de la Cruz F. J. Mol. Biol. 2006; 358: 857-869Crossref PubMed Scopus (62) Google Scholar) relaxase with bound ssDNA, the Asp a bond with the of the second HUH the distance is whereas for the relaxase is A.F. Ozburn A. Xia S. Meyer R.J. Robertus J.D. J. Mol. Biol. PubMed Scopus Google Scholar), the distance between a and the is a bond with is in or The distance of from may the of metal that the metal the transfer In F the a bond with the of the second tyrosine of the YY-X5-6-YY motif that this family of bacterial relaxases (15Koonin E.V. Ilyina T.V. Biosystems. 1993; 30: 241-268Crossref PubMed Scopus (152) Google Scholar). The role of this Tyr in the cleavage mechanism is (12Street L.M. Harley M.J. Stern J.C. Larkin C. Williams S.L. Miller D.L. Dohm J.A. Rodgers M.E. Schildbach J.F. Biochim. Biophys. Acta. 2003; 1646: 86-99Crossref PubMed Scopus (38) Google Scholar) and that reduced in vitro activity with both and the of for is near at in vivo activity is transfer is reduced to The that may in a network that the scissile however, the that a by the structures to directly in cleavage. His in is a metal Asp or T. C. Chem. 2003; PubMed Scopus Google Scholar). coordination by His a the of the scissile phosphate and the to by the catalytic The reduced transfer of the and are consistent with reduced relaxase activity to of the are for the reduced activity of the reduced affinity for ssDNA and reduced affinity for to and L.M. Ragonese H. Matson S.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) a the for the the bound metal. was results from TraI in vitro and was in is however, is the of acidic at and in the the HUH motif and metal coordination are conserved in the His residues are by the motif in the and relaxases related to A. Lucas M. C. de la Cruz F. Mol. Microbiol. 2003; PubMed Scopus Google Scholar). that the motif is important for function. of the motif by His residues a protein that facilitated transfer at a The reduced function of the that the protein is for of the The between and the HUH relaxases required to efficient of the motif have the His residues that the metal contribute to TraI relaxase the transfer results of the that His residues are important to TraI function. a bond to is essential to function. The network of residues in relaxase structures that to bind a to in the relaxase a relatively the must the scissile the Tyr the and the residues that bind the metal. The of the His and the interacting Asp a affinity for metal without affinity for ssDNA by placing an acidic amino near a ssDNA the of His residues for binding of the metal while the of the of the may be for cleavage TraI is to the motif to bind metal the protein a single metal in its cleavage that a reaction mechanism acidic residues to metal is required to the of the however, other HUH as and a single metal in cleavage reaction have an acidic residue in metal the cleavage of the TraI further to in transfer of and the conjugative plasmid. and be to these