Functionally Significant Mobile Regions of Escherichia coli SecA ATPase Identified by NMR
2002; Elsevier BV; Volume: 277; Issue: 52 Linguagem: Inglês
10.1074/jbc.m209237200
ISSN1083-351X
AutoresYi-Te Chou, Joanna F. Swain, Lila M. Gierasch,
Tópico(s)Protein Structure and Dynamics
ResumoSecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the cytoplasmic membrane once bound to both the signal sequence and portions of the mature domain of the preprotein. SecA is proposed to undergo major conformational changes during translocation. These conformational changes are accompanied by major rearrangements of SecA structural domains. To understand the interdomain rearrangements, we have examined SecA by NMR and identified regions that display narrow resonances indicating high mobility. The mobile regions of SecA have been assigned to a sequence from the second of two domains with nucleotide-binding folds (NBF-II; residues 564–579) and to the extreme C-terminal segment of SecA (residues 864–901), both of which are essential for preprotein translocation activity. Interactions with ligands suggest that the mobile regions are involved in functionally critical regulatory steps in SecA. SecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the cytoplasmic membrane once bound to both the signal sequence and portions of the mature domain of the preprotein. SecA is proposed to undergo major conformational changes during translocation. These conformational changes are accompanied by major rearrangements of SecA structural domains. To understand the interdomain rearrangements, we have examined SecA by NMR and identified regions that display narrow resonances indicating high mobility. The mobile regions of SecA have been assigned to a sequence from the second of two domains with nucleotide-binding folds (NBF-II; residues 564–579) and to the extreme C-terminal segment of SecA (residues 864–901), both of which are essential for preprotein translocation activity. Interactions with ligands suggest that the mobile regions are involved in functionally critical regulatory steps in SecA. nucleotide-binding fold heteronuclear single quantum correlation intramolecular regulator of ATP hydrolysis nuclear Overhauser effect NOE spectroscopy 3-(trimethylsilyl) propionate-2,2,3,3-d4 total correlation spectroscopy wild-type Preprotein translocation in bacterial cells is driven by SecA, a dissociable peripheral membrane ATPase that recognizes the protein precursor and assists its translocation across the inner membrane (1Oliver D.B. Mol. Microbiol. 1993; 7: 159-165Crossref PubMed Scopus (75) Google Scholar, 2Duong F. Eichler J. Price A. Leonard M.R. Wickner W. Cell. 1997; 91: 567-573Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 3Driessen A.J. Fekkes P. van der Wolk J.P. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (148) Google Scholar, 4Danese P.N. Silhavy T.J. Annu. Rev. Genet. 1998; 32: 59-94Crossref PubMed Scopus (189) Google Scholar, 5Economou A. Mol. Microbiol. 1998; 27: 522-528Crossref Scopus (65) Google Scholar). Profound conformational changes are necessary for SecA to alternately bind to the preprotein/SecB complex, associate with the membrane/SecYEG translocase channel, and drive the stepwise translocation of the preprotein across the membrane. These steps are coordinated by ATP binding and hydrolysis (6Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (465) Google Scholar, 7Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 8Breukink E. Nouwen N. van Raalte A. Mizushima S. Tommassen J. de Kruijff B. J. Biol. Chem. 1995; 270: 7902-7907Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 9van der Wolk J.P. de Wit J.G. Driessen A.J.M. EMBO J. 1997; 16: 7297-8304Crossref PubMed Scopus (161) Google Scholar). SecA conformational changes appear to be achieved by as-yet poorly understood rearrangement of multiple independently folded domains of SecA. The domain structure of SecA was originally suggested by limited proteolysis, which yields reproducible patterns of resistant fragments (10Triplett T.L. Sgrignoli A.R. Gao F. Yang Y. Tai P.C. Gierasch L.M. J. Biol. Chem. 2001; 276: 19648-19655Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 11Eichler J. Wickner W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5574-5581Crossref PubMed Scopus (73) Google Scholar, 12Eichler J. Brunner J. Wickner W. EMBO J. 1997; 16: 2188-2196Crossref PubMed Scopus (57) Google Scholar, 13Song M. Kim H. J. Biol. Chem. 1997; 122: 1010-1018Google Scholar, 14Price A. Economou A. Duong F. Wickner W. J. Biol. Chem. 1996; 271: 31580-31584Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 16Sianidis G. Karamanou S. Vrontou E. Boulias K. Repanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 2001; 20: 961-970Crossref PubMed Scopus (95) Google Scholar, 17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 18Dempsey B.R. Economou A. Dunn S.D. Shilton B.H. J. Mol. Biol. 2002; 315: 831-843Crossref PubMed Scopus (26) Google Scholar, 19Dapic V. Oliver D. J. Biol. Chem. 2000; 275: 25000-25007Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Nakatogawa H. Mori H. Ito K. J. Biol. Chem. 2000; 275: 33209-33212Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Moreover, several of these fragments have been expressed as individual domains (18Dempsey B.R. Economou A. Dunn S.D. Shilton B.H. J. Mol. Biol. 2002; 315: 831-843Crossref PubMed Scopus (26) Google Scholar, 19Dapic V. Oliver D. J. Biol. Chem. 2000; 275: 25000-25007Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Nakatogawa H. Mori H. Ito K. J. Biol. Chem. 2000; 275: 33209-33212Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The recent crystal structure of the cytoplasmic form of Bacillus subtilis SecA (21Hunt J.F. Weinkauf S. Henry L. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar) provides structural insights into the domain folds and interdomain packing and confirmed that this large protein is poised to undergo major domain rearrangements as it performs its multiple functions. Within the N-terminal domain are two nucleotide-binding folds, termed NBF-I1 and NBF-II, which exhibit high homology to RNA and DNA helicases from superfamilies 1 and 2 (22Caruthers J.M. McKay D.B. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (453) Google Scholar). Furthermore, as predicted from sequence data (23Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1032) Google Scholar), all of the expected DEAD-box helicase motifs (I through VI) are found in structurally homologous locations in SecA NBF-I and -II. As in the helicases, the nucleotide-binding site is at the NBF-I/NBF-II interdomain junction, and the conserved motifs juxtapose the binding site. A novel insertion in NBF-I (residues 220–380, Escherichia coli numbering), not present in helicases, was found in an early study of E. coli SecA to cross-link a precursor protein (24Kimura E. Akita M. Matsuyama S. Mizushima S. J. Biol. Chem. 1991; 266: 6600-6606Abstract Full Text PDF PubMed Google Scholar) and therefore is termed the preprotein cross-linking domain. The last 39 residues at the C terminus of SecA are disordered and not resolved in the crystal structure. Structures of DEAD-box helicases suggest that their mechanisms rely on regulated rigid body rearrangements of the two nucleotide-binding domains that require interdomain flexibility (22Caruthers J.M. McKay D.B. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (453) Google Scholar). Consistent with the structural similarity between SecA and the helicases, numerous published data support the importance of interdomain interactions in the SecA mechanism. Schmidt et al. (25Schmidt M. Ding H. Ramamurthy V. Mukerji I. Oliver D. J. Biol. Chem. 2000; 275: 15440-15448Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) reported that several suppressors of protein secretion defects and azide-resistant mutants of SecA perturbed the SecA endothermic transition by weakening interdomain contacts. Several laboratories, including our own, have shown that any disruption of the C terminus of SecA, through truncation or partial unfolding, leads to enhanced ATPase activity (10Triplett T.L. Sgrignoli A.R. Gao F. Yang Y. Tai P.C. Gierasch L.M. J. Biol. Chem. 2001; 276: 19648-19655Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Song M. Kim H. J. Biol. Chem. 1997; 122: 1010-1018Google Scholar, 14Price A. Economou A. Duong F. Wickner W. J. Biol. Chem. 1996; 271: 31580-31584Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Deletion of a specific region of the C-terminal sequence (residues 783 to 795) recapitulates the ATPase activation observed in C-terminal truncated versions of SecA; this region has been termed by Economou and coworkers (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar) the intramolecular regulator of ATP hydrolysis 1 (IRA1). These same researchers presented biochemical evidence that a region nearly coincident with NBF-II (amino acids 462 to 610, dubbed IRA2) regulates the nucleotide binding and release function of NBF-I (16Sianidis G. Karamanou S. Vrontou E. Boulias K. Repanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 2001; 20: 961-970Crossref PubMed Scopus (95) Google Scholar). Recent cross-linking and biochemical experiments by Economou and coworkers (17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) suggest that binding of signal peptides occurs to a segment from residues 219 to 244 and that IRA1 regulates access to this binding site. Binding of SecB to the C-terminal region of SecA enhances its ATPase activity and promotes exchange with membrane-bound SecA (26Woodbury R.L. Topping T.B. Diamond D.L. Suciu D. Kumamoto C.A. Hardy S.J. Randall L.L. J. Biol. Chem. 2000; 275: 24191-24198Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition to the domain reorganization within a SecA monomer, binding to phospholipids or signal peptides modulates SecA oligomerization with lipids favoring monomeric states and signal peptides shifting the equilibrium toward dimers (27Benach, J., Chou, Y.-T., Fak, J. J., Itkin, A., Nicolae, D. D., Smith, P. C., Wittrock, G., Floyd, D., Gierasch, L. M., and Hunt, J. F. (October 27, 2002) J. Biol. Chem. 10.1074/jbc.M205992200Google Scholar). Clearly, a delicate balance of interdomain interactions poises SecA to undergo profound conformational change in a regulated way, depending upon binding to its many ligands. The ligand-modulated changes in interdomain packing in SecA necessarily require regions of sequence that are able to adopt alternate conformations in order to allow rigid body rearrangements. It is reasonable to anticipate that some regions of SecA will show enhanced mobility, enabling them to perform regulatory or linker roles in its mechanistic cycle. Static pictures of the SecA structure do not reveal the locations and dynamics of these functionally important regions. Therefore, we embarked on an effort using NMR to ask whether there are mobile regions in SecA and to understand their roles in its domain rearrangements. This approach had been productive in our previous work on GroES, where the GroEL-interactive regions emerged as ‘mobile loops’ that displayed narrow resonances in both one- and two-dimensional NMR spectra (28Landry S.J. Zeilstra-Ryalls J. Fayet O. Georgopoulos C. Gierasch L.M. Nature. 1993; 364: 255-258Crossref PubMed Scopus (212) Google Scholar). In examination of SecA, we indeed found several narrow resonances in NMR spectra, and we have been able to carry out sequence-specific assignment of these resonances. Strikingly, these regions have been assigned to residues within NBF-II (mobile region 1, residues 564 to 579) and the C terminus (mobile region 2, residues 864 to 901), both of which play critical roles in SecA function and both of which are structurally related to the proposed IRA regions. Unless specifically mentioned, standard laboratory reagents were purchased from Sigma or VWR. All deuterated or15N-labeled compounds were purchased from Cambridge Isotope Laboratories (Andover, MA) or Isotec (Miamisburg, OH). SecA and variants were purified from overexpressing E. coli strains BL21.19 (pT7-SecA2, pT7-SecA95) kindly provided by D. Oliver (Wesleyan University) (29Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar,30Rajapandi T. Oliver D. Biochem. Biophys. Res. Commun. 1994; 200: 1477-1483Crossref PubMed Scopus (31) Google Scholar). All proteins were purified as described previously (10Triplett T.L. Sgrignoli A.R. Gao F. Yang Y. Tai P.C. Gierasch L.M. J. Biol. Chem. 2001; 276: 19648-19655Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) with minor modifications. Uniform 15N-labeled SecA protein was obtained by growing cells in M9 minimal medium containing15NH4Cl. The sample with reverse selective labeling of arginine residues was prepared by providing cells with 250 mg/liter culture of unlabeled arginine in addition to15NH4Cl. Purity of SecA was assessed by SDS-PAGE; samples of lower than 98% purity were found to aggregate during concentration. The principal impurities observed were proteolytic breakdown products (molecular masses 95, 75, 64, 43 kDa), most of which are less soluble than the intact protein. ATPase activity of purified SecA was verified to be comparable with literature values (10Triplett T.L. Sgrignoli A.R. Gao F. Yang Y. Tai P.C. Gierasch L.M. J. Biol. Chem. 2001; 276: 19648-19655Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Proteins were concentrated using Centricon-30 or -50 (Millipore) to achieve millimolar concentration for NMR. Protein concentrations were measured via the Bradford assay. At least three individual preparations of fresh protein samples were used to confirm the reproducibility of the data. Samples for NMR spectrometry consisted of 0.8–1.2 mm SecA (monomer) in 25 mm KCl, 25 mm potassium phosphate buffer (pH 7.0–7.7), and 10% D2O with or without 0.02% azide. Under these conditions, SecA is overwhelmingly in the dimer state (31Woodbury R.L. Hardy S.J.S. Randall L.L. Protein Sci. 2002; 11: 875-882Crossref PubMed Scopus (125) Google Scholar).2 Spectra were obtained using Bruker AMX500 or Avance 600 NMR spectrometers at 25 °C. 1H chemical shifts were referenced with respect to the methyl protons of 3-(trimethylsilyl) propionate (TMSP) (0 ppm). Data were processed within the FELIX 97 program (Biosym Technologies, San Diego) running on a Silicon Graphics Indigo work station (Mountain View, CA).1H-15N HSQC experiments were recorded using the States-TPPI method (32Cavanagh J. Fairbrother W.J. Palmer III, A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego1996Google Scholar) with 32–64 scans; the spectral widths and number of points were 7000–8000 Hz × 1650–4000 Hz and 768 × 128 points in the 1H and 15N dimensions, respectively. The spectral widths and number of complex points in the F3, F2, and F1 dimensions, the number of scans per free induction decay, and the total measurement time of three-dimensional1H-15N TOCSY-HSQC and1H-15N NOESY-HSQC (32Cavanagh J. Fairbrother W.J. Palmer III, A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego1996Google Scholar) were 7000 × 1650 × 7000 Hz, 512 × 32 × 64, and 16 scans for ∼5 days. The mixing times were 29 ms for the TOCSY-HSQC and 70 ms for the NOESY-HSQC experiments. As a 204-kDa homodimer, SecA is beyond the size of proteins routinely assignable via NMR methods. A roughly spherical protein of M r 200 would typically be expected to have broad signals (>100 Hz), consistent with an overall correlation time (τc) of ∼100 ns (32Cavanagh J. Fairbrother W.J. Palmer III, A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego1996Google Scholar). This protein, however, contains a set of relatively narrow peaks superimposed on an envelope of broad, overlapping resonances in the one-dimensional 1H and two-dimensional 1H-15N HSQC NMR spectra (Fig. 2). The narrow resonances indicate that this protein contains some significantly mobile residues. We localized the amino acid sequences corresponding to these resonances of SecA using methods of sequential assignment (32Cavanagh J. Fairbrother W.J. Palmer III, A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego1996Google Scholar, 33Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley and Sons, New York1986Crossref Google Scholar), relying on1H-15N HSQC, 1H-15N TOCSY-HSQC, and 1H-15N NOESY-HSQC data obtained in 90% H2O/10% D2O on uniformly15N-labeled samples. The assignment was initiated by identifying unique spin systems (e.g. serine, threonine, alanine, glycine, and others) in the three-dimensional TOCSY data. Sequential connections between those specific residues or other spin systems (e.g. S-AMX, AMX-G, S-A, T-G, and S-G) were identified in the heteronuclear NOESY spectrum. Further sequential assignments of backbone amide resonances were tentatively made by additional NOEs extracted from the three-dimensional NOESY-HSQC data. We confirmed and extended our assignments using “reverse selective labeling of arginine” (34Rizo J. Liu Z.-P. Gierasch L.M. J. Biomol. NMR. 1994; 4: 741-760Crossref PubMed Scopus (38) Google Scholar), in which all residues but arginine are15N-labeled (Fig. 1 D, locations of assigned arginines indicated by open circles).Figure 1Assignment of two mobile regions in E. coli SecA by NMR. A, sequences of the two mobile regions of E. coli SecA; residues in color (red for mobile region 1 and blue for mobile region 2) could be assigned as described under “Results.”B, representative slices from the1H-15N NOESY-HSQC spectrum corresponding to residues Gly888 to Lys894; sequential connectivities indicated by horizontal lines. C, two-dimensional 1H-15N HSQC spectrum of uniformly 15N-labeled wt SecA showing definitive assignments of cross peaks corresponding to backbone amide protons (mobile region 1 in red and mobile region 2 inblue; side chain amides of Asn and Gln in boxed region). D, reverse selective labeling of Arg residues in wt SecA (non-Arg-labeled 1H-15N HSQC):open circles correspond to positions of cross peaks of arginine residues observed in uniformly 15N-labeled wt SecA spectra but missing when Arg is unlabeled.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Based on all NMR data, we were able to determine that the mobile resonances of E. coli SecA correspond to amino acids 564 to 579 (mobile region 1) and 864 to 901 (mobile region 2) (Fig. 1 A). The following example illustrates the approach used: The assignment of mobile region 1 was initiated by unique assignment of570QLRG573. Sequential Hα-HN, HN-HN, and Hβ-HN NOEs were used to define a Glu/Gln/Met-Leu/Ile-Arg sequence. In this case, the third residue was shown to be arginine by reverse labeling; other residue identifications were obtained from 1H-15N TOCSY-HSQC data. Search of the SecA sequence for a region consistent with these data yields two possible assignments (418MIR420 and570QLR572). A strong Hα-HN NOE between the arginine and an i+1 glycine residue uniquely identifies these resonances as the 570QLRG573 sequence. Another set of resonances correlated by Hα-HN, HN-HN, and Hβ-HN sequential NOEs consists of an arginine followed by a Lys/Glu/Gln/Met spin system, which itself has an Hα-HN NOE to an i+1 glycine residue. Only two regions of the SecA sequence fit these criteria (577RQG579 and786RQG788). Here and throughout this assignment procedure, we have made the plausible assumption that short segments of mobile residues separated by a short unassignable sequence in the context of a 901-amino acid dimer arise from a single, larger mobile region. For example, we found no other mobile residues in the vicinity of 786RQG788 and hence assigned this RQG sequence to residues 577–579. Similar logic allowed us to tentatively assign a Ser-Arg pair and an Arg-Ile/Leu pair to the564SRRI567 sequence. Thus we conclude that one mobile region comprises residues Ser564 to Gly579 (mobile region 1). In an analogous fashion, we assigned mobile region 2 to the extreme C terminus of the SecA protein (residues 864 to 901). Assigned sequences in this region included 864SA865,870LAAGTGER877,879VGRN882,888GSGKKYKQCHGRLQ901. We argue that this is one consecutive mobile region based on similar assumptions to those used for mobile region 1. As shown in Fig. 2, the HSQC spectrum of SecA upon addition of either Mg2+ alone or Mg/ATP-γS reveals significant chemical shift perturbation (both proton and nitrogen dimensions) to selected resonances, notably Arg566 and Arg572 in mobile region 1. Mg2+ is reported to bind to SecA directly, with its proposed site of binding involving Asp209 and Asp217 (E. colinumbering) (35van der Wolk J.P.W. Klose M. de Wit J.G. den Blaauwen T. Freudl R. Driessen A.J.M. J. Biol. Chem. 1995; 270: 18975-18982Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The former residue is part of the DEAD-box sequence of SecA (helicase motif II), 209DEVD212, the last residue of which interacts directly with Gln570 of helicase motif VI (and mobile region I) in the structures of both SecA (21Hunt J.F. Weinkauf S. Henry L. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar) and UvrB (36Machius M. Henry L. Palnitkar M. Deisenhofer J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11717-11722Crossref PubMed Scopus (94) Google Scholar, 37Theis K. Chen P.J. Skorvaga M. van Houten B. Kisker C. EMBO J. 1999; 18: 6899-6907Crossref PubMed Scopus (165) Google Scholar). Asp217 directly interacts with Arg566 of mobile region 1. The involvement of helicase motif VI in forming the nucleotide-binding site is consistent with the sensitivity of Gln570, Arg572, and Arg577 to addition of ATP-γS. Interestingly, in addition, significant chemical shift changes are observed for Arg566in mobile region 1 when ATP-γS binds, even though this residue does not directly contact the nucleotide, and for Lys894 and Lys891 in mobile region 2, the latter upon binding of ATP-γS but not Mg2+. Further insight into the spatial arrangement of the mobile regions was obtained through the use of Mn2+ ion as a paramagnetic broadening agent. Nuclei within ∼20 Å of the site of Mn2+ binding may be perturbed, and the effect is distance-dependent by an inverse third power (38Mildvan A.S. Engle J.L. Methods Enzymol. 1972; 26: 654-682Crossref PubMed Scopus (87) Google Scholar, 39Dwek R.A. NMR in Biochemistry. Clarendon Press, Oxford1973Google Scholar, 40Grisham C.M. 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Additionally, the signal from an internal standard (TMSP) was not broadened (data not shown), supporting the interpretation that the observed changes arise from specific binding. Because Mn2+ functionally substitutes for Mg2+in NTP binding to most ATPases and GTPases (42Wittinghofer A. Pai E.F. Trends Biochem. Sci. 1991; 16: 382-387Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 43Schweins T. Scheffzek K. Assheuer R. Wittinghofer A. J. Mol. Biol. 1997; 266: 847-856Crossref PubMed Scopus (52) Google Scholar), we could explore nucleotide-induced domain rearrangements of SecA with the added Mn2+ reporting on the location of mobile regions 1 and 2 with respect to the nucleotide-binding site. Surprisingly, many (but not all) 1H-15N cross peaks in the HSQC spectrum arising from the mobile regions were restored to nearly their original intensity when AMP-PNP was added (Fig. 3 C), arguing that AMP-PNP reversed the influence of Mn2+. As indicated above, the observation that AMP-PNP restored the intensities of some, but not all, broadened signals points to a specific effect of this ligand. Our results reveal that there are two well defined regions of high mobility in SecA, such that narrow NMR resonances are observable despite the large size of this protein. Moreover, the locations of these regions are functionally provocative. Thus, SecA joins several multidomain proteins that have been found to have high mobility in functionally important regions (28Landry S.J. Zeilstra-Ryalls J. Fayet O. Georgopoulos C. Gierasch L.M. Nature. 1993; 364: 255-258Crossref PubMed Scopus (212) Google Scholar, 44Jasanoff A. Park S.J. Wiley D.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9900-9904Crossref PubMed Scopus (40) Google Scholar, 45Liang H. Petros A.M. Meadows R.P. Yoon H.S. Egan D.A. Walter K. Holzman T.F. Robins T. Fesik S.W. Biochemistry. 1996; 35: 2095-2103Crossref PubMed Scopus (59) Google Scholar, 46Volkert T.L. Baleja J.D. Kumamoto C.A. Biochem. Biophys. Res. Commun. 1999; 264: 949-954Crossref PubMed Scopus (20) Google Scholar). Mobile region 1 of SecA spans amino acids 564 to 579 and contains the conserved helicase motif VI (570–577) (23Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1032) Google Scholar). This region has direct contact with bound nucleotide in several helicases (Ref. 22Caruthers J.M. McKay D.B. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (453) Google Scholar and references within), and the recent crystal structure of B. subtilis SecA confirms that the same role is played in SecA (21Hunt J.F. Weinkauf S. Henry L. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar). Alignment of 30 different SecA sequences shows it to be one of the most conserved sequences in SecA (near 100%) (21Hunt J.F. Weinkauf S. Henry L. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar). Several mutations that are within or near mobile region 1 cause altered SecA ATPase activity (16Sianidis G. Karamanou S. Vrontou E. Boulias K. Repanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 2001; 20: 961-970Crossref PubMed Scopus (95) Google Scholar, 20Nakatogawa H. Mori H. Ito K. J. Biol. Chem. 2000; 275: 33209-33212Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The second mobile segment (mobile region 2) consists of the extreme 43 C-terminal residues of SecA, a region that has been shown to be important for several functions: translocation activity in vivo, lipid binding, SecB binding, and Zn2+ binding (8Breukink E. Nouwen N. van Raalte A. Mizushima S. Tommassen J. de Kruijff B. J. Biol. Chem. 1995; 270: 7902-7907Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 30Rajapandi T. Oliver D. Biochem. Biophys. Res. 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Chem. 2000; 275: 24191-24198Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) have suggested that this C-terminal region is not only a SecB-binding site but that the interaction between SecB and the C terminus of SecA is also crucial to trigger the conversion from the inactive to the active form of the SecA/SecB complex, promoting exchange with membrane-bound SecA. In addition, Kendall and coworkers (52Kim J. Miller A. Wang L.
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