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Lys13 Plays a Crucial Role in the Functional Adaptation of the Thermophilic Triose-phosphate Isomerase fromBacillus stearothermophilus to High Temperatures

1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês

10.1074/jbc.274.27.19181

ISSN

1083-351X

Autores

Marco Alvarez, Johan Wouters, Dominique Maes, Véronique Mainfroid, Françoise Rentier‐Delrue, Lode Wyns, Eric Depiereux, Joseph Martial,

Tópico(s)

Protein Structure and Dynamics

Resumo

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) andThermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the “mesophilic residues” Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-Å resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption. The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) andThermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the “mesophilic residues” Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-Å resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption. triose-phosphate isomerase TIM of T. maritima and B. stearothermophilus, respectively polymerase chain reaction 4-morpholineethanesulfonic acid root mean square Understanding the molecular adaptation of proteins to extreme temperatures remains a major challenge for protein scientists. Over the last 2 decades, several studies have attempted to establish a general rule leading to thermostability, but no overall consensus explanation could be formulated (1Jaenicke R. Schurig H. Beaucamp N. Ostendorp R. Adv. Protein Chem. 1996; 48: 181-269Crossref PubMed Google Scholar). For example, although the composition of thermophilic proteins shows that certain amino acids are preferred to ones present in mesophilic homologues, no general trends emerge. Studies have demonstrated the need to place the analysis of primary structure in a three-dimensional structural context (2Russel R.J. Tylor G.L. Curr. Opin. Biotechnol. 1995; 6: 370-374Crossref PubMed Scopus (106) Google Scholar); structural determinants such as ionic networks (3Rice D.W. Yip K.S. Stillman T.J. Britton K.L. Fuentes A. Connerton I. Pasquo A. Scandura R. Engel P.C. FEMS Microbiol. Rev. 1996; 18: 105-117Crossref PubMed Google Scholar, 4Pfeil W. Gesierich U. Kleeman G.R. Sterner R. J. Mol. Biol. 1997; 272: 591-596Crossref PubMed Scopus (63) Google Scholar), hydrophobic packing (5Peters J. Baumeister W. Lupas A. J. Mol. Biol. 1996; 257: 1031-1041Crossref PubMed Scopus (92) Google Scholar, 6Hennig M. Sterner R. Kirschner K. Jansonius J.N. Biochemistry. 1997; 36: 6009-6016Crossref PubMed Scopus (90) Google Scholar), and cooperative associations (5Peters J. Baumeister W. Lupas A. J. Mol. Biol. 1996; 257: 1031-1041Crossref PubMed Scopus (92) Google Scholar, 7Lim J.H. Yu Y.G. Han Y.S. Cho S. Ahn B.Y. Kim S.H. Cho Y. J. Mol. Biol. 1997; 270: 259-274Crossref PubMed Scopus (116) Google Scholar) seem to better reflect the general features associated with thermal adaptation. All of them, individually or in combination, enable thermophilic proteins to adapt to extreme temperatures. Thermotoga maritima is a hyperthermophilic eubacterium of the order Thermotogales, the oldest branch within the bacterial domain. Its optimal growth temperature is 80 °C (8Huber R. Langworthy T.A. König H. Thomm M. Woese C.R. Sleytr U.B. Stetter K.O. Arch. Microbiol. 1986; 144: 324-333Crossref Scopus (633) Google Scholar). Bacillus stearothermophilus is a moderately thermophilic endospore-forming Gram-positive rod growing optimally at about 65 °C (9Fahey R.C. Kalb E. Harris J.I. Biochem. J. 1971; 124: 77PCrossref PubMed Google Scholar). Several glycolytic pathway enzymes from these strains have been produced by recombinant DNA technology, and all of them display optimal catalytic activity at temperatures above the optimal growth temperature of the source organism (1Jaenicke R. Schurig H. Beaucamp N. Ostendorp R. Adv. Protein Chem. 1996; 48: 181-269Crossref PubMed Google Scholar). Triose-phosphate isomerase (TIM)1 is one of them. TIM is a homodimer catalyzing the interconversion of dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate in the glycolytic pathway (10Rieder S.V. Rose I.A. J. Biol. Chem. 1959; 234: 1007-1010Abstract Full Text PDF PubMed Google Scholar). It is the prototype of a family comprising many different members sharing a common folding motif called the α/β barrel. This fold consists of eight parallel β-strands forming a barrel, surrounded by eight α-helices (11Farber G.K. Petsko G.A. Trends Biochem. Sci. 1990; 15: 228-234Abstract Full Text PDF PubMed Scopus (420) Google Scholar). The active site of TIM lies at the carboxyl-terminal end of the β-barrel, and the catalytic residues belong to loops connecting the β-strands to the following α-helices (Lys10, His94, and Glu166; B. stearothermophilus TIM numbering). At present, the three-dimensional structures of 11 TIMs are known: those of chicken (12Banner D.W. Bloomer A.C. Petsko G.A. Philips D.C. Pogson C.I. Wilson I.A. Corran P.H. Furth A.J. Milman J.D. Offord R.E. Priddle J.D. Waley S.G. Nature. 1975; 255: 609-614Crossref PubMed Scopus (600) Google Scholar), yeast (13Lolis E. Alber T. Davenport R.C. Rose D. Hartman F.C. Petsko G.A. Biochemistry. 1990; 29: 6609-6618Crossref PubMed Scopus (254) Google Scholar), Trypanosoma brucei (14Wierenga R.K. Noble M.E.M. Vriend G. Nauche S. Hol W.G.J. J. Mol. Biol. 1991; 220: 995-1015Crossref PubMed Scopus (163) Google Scholar),Escherichia coli (15Noble M.E.M. Zeelen J.P. Wierenga R.K. Mainfroid V. Goraj K. Gohimont A.-C. Martial J.A. Acta Crystallogr. Sec. D. 1993; 49: 403-417Crossref PubMed Google Scholar), human (16Mande S.C. Mainfroid V. Kalk K.H. Goraj K. Martial J.A. Hol W.G.J. Protein Sci. 1994; 3: 810-821Crossref PubMed Scopus (124) Google Scholar), B. stearothermophilus (17Delboni L.F. Mande S.C. Rentier-Delrue F. Mainfroid V. Turley S. Vellieux M.D. Martial J.A. Hol W.G.J. Protein Sci. 1995; 4: 2594-2604Crossref PubMed Scopus (114) Google Scholar), Plasmodium falciparum (18Velanker S.S. Ray S.S. Gokhale R.S. Hemalatha Balaram S.S. Murthy M.R.N. Structure. 1997; 5: 751-761Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar),Vibrio marinus (19Alvarez M. Zeelen J.P. Mainfroid V. Rentier-Delrue F. Martial J.A. Wyns L. Wierenga R.K. Maes D. J. Biol. Chem. 1998; 273: 2199-2206Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), Trypanosoma cruzei (20Maldonado E. Soriano-Garcia M. Moreno A. Cabrera N. Garza-Ramos G. Tuena de Gomez-Puyou M. Gomez-Puyou A. Perez-Monfort R. J. Mol. Biol. 1998; 283: 193-203Crossref PubMed Scopus (63) Google Scholar),Leishmania mexicana (21Williams J.C. Zeelen J.Ph. Neubauer G. Vriend G. Backmann J. Michels P.A.M. Lambeir A.-M. Wierenga R.K. Protein Eng. 1999; 12: 243-250Crossref PubMed Scopus (106) Google Scholar), and T. maritima. 2D. Maes, J. P. Zeelen, N. Thanki, N. Beaucamp, M. Alvarez, M. H. Dao-Thi, J. Backmann, J. A. Martial, L. Wyns, R. Jaenicke, and R. K. Wierenga, manuscript in preparation. B. stearothermophilus TIM (bTIM) is reported to display moderately thermophilic enzymatic properties (22Rentier-Delrue F. Mande S.C. Moyens S. Terpstra P. Mainfroid V. Goraj K. Hol W.G.J. Martial J.A. J. Mol. Biol. 1993; 229: 85-93Crossref PubMed Scopus (75) Google Scholar). Resolution of its crystallographic structure has made it possible to propose certain features as responsible for its increased thermostability, such as higher content of proline residues at the N1 position in helices, smallest number and volume of cavities, and more buried hydrophobic surfaces upon dimerization than E. coli TIM (17Delboni L.F. Mande S.C. Rentier-Delrue F. Mainfroid V. Turley S. Vellieux M.D. Martial J.A. Hol W.G.J. Protein Sci. 1995; 4: 2594-2604Crossref PubMed Scopus (114) Google Scholar). T. maritima TIM (tTIM), described by Schurig et al. (23Schurig H. Beaucamp N. Ostendorp R. Jaenicke R. Adler E. Knowles J.R. EMBO J. 1995; 14: 442-451Crossref PubMed Scopus (69) Google Scholar), is an extremely thermophilic protein resistant to temperatures above 90 °C. Surprisingly, it is produced as a fusion protein jointly with phosphoglycerate kinase, another glycolytic enzyme. Recently, it has been proposed that the tetrameric quaternary structure adopted by the polyprotein permits conservation of the dimeric form necessary for TIM activity and increases slightly the thermal stability of tTIM (24Beaucamp N. Hofmann A. Kellerer B. Jaenicke R. Protein Sci. 1997; 6: 2159-2165Crossref PubMed Scopus (46) Google Scholar). Although many TIM sequences have been reported, few of these proteins have been fully characterized in terms of thermal stability. Alignment of these sequences reveals a particular pair substitution in one of the conserved regions of “thermophilic TIMs” (bTIM and tTIM), an N12H/G13K replacement. Residues 12 and 13 belong to a region of variable length named loop 1, joining strand β1 to helix α1 and located at the TIM dimer interface. The presence of the His12-Lys13 pair is also present in the TIM of a Synechocystis sp. strain, a mesophilic cyanobacterium (25Kaneko T. Tanaka A. Sato S. Kotani H. Sazuka T. Miyajima N. Sugiura M. Tabata S. DNA Res. 1995; 2: 153-166Crossref PubMed Scopus (263) Google Scholar). His12 has been found in the TIMs of Pseudomonas syringae 3J. J. Rich and D. K. Willis, nucleotide sequence of Pseudomonas syringae TIM, ID TPIS-PSESY, Swiss-Prot Data Bank code (AC) P95576. andBorrelia burgdorferi (27Gebbia J.A. Backenson P.B. Coleman J.L. Anda P. Benach J.L. Gene (Amst.). 1997; 188: 221-228Crossref PubMed Scopus (21) Google Scholar), and Lys13 has been found in Bacillus subtilis TIM (28Leyva-Vazquez M.A. Setlow P. J. Bacteriol. 1994; 176: 3903-3910Crossref PubMed Google Scholar), but there is no available information on the enzymatic properties and stability of these TIMs. The high conservation of Asn12 in most TIMs can be explained by the ability of the Asn side chain amide nitrogen to interact with residues Trp9 and Met11 of the same loop, thereby stabilizing the catalytic Lys10 of the active site (17Delboni L.F. Mande S.C. Rentier-Delrue F. Mainfroid V. Turley S. Vellieux M.D. Martial J.A. Hol W.G.J. Protein Sci. 1995; 4: 2594-2604Crossref PubMed Scopus (114) Google Scholar). At high temperature, an Asn residue at the edge of the protein's dimeric interface (partially solvent-exposed) is susceptible to deamidation leading to irreversible heat inactivation of the enzyme (29Yuan P.M. Talent J.M. Gracy R.W. Mech. Ageing Dev. 1981; 17: 151-162Crossref PubMed Scopus (70) Google Scholar). In solution, deamidation occurs through intra- molecular nucleophilic attack by the backbone amide nitrogen on the γ-carbonyl carbon of the Asn side chain, resulting in conversion of Asn residues to aspartate or isoaspartate and release of ammonia (30Tyler-Cross R. Schirch V. J. Biol. Chem. 1991; 266: 22549-22556Abstract Full Text PDF PubMed Google Scholar). The rate of deamidation in proteins depends on the pH, ionic strength, temperature, sequence, and structure (30Tyler-Cross R. Schirch V. J. Biol. Chem. 1991; 266: 22549-22556Abstract Full Text PDF PubMed Google Scholar, 31Tomazic S.J. Klibanov A.M. J. Biol. Chem. 1988; 263: 3086-3091Abstract Full Text PDF PubMed Google Scholar, 32Hayes C.S. Setlow P. J. Bacteriol. 1997; 179: 6020-6027Crossref PubMed Google Scholar). Replacement of Asn residues is used in protein engineering to thermostabilize proteins: inAspergillus awamori glucoamylase, for instance, it was possible to increase its thermostability severalfold (33Chen H. Ford C. Reilly P.J. Biochem. J. 1994; 301: 275-281Crossref PubMed Scopus (65) Google Scholar). Furthermore, Asn residues followed by Gly appear considerably more sensitive to deamidation (34Kossiakoff A.A. Science. 1988; 240: 191-194Crossref PubMed Scopus (172) Google Scholar, 35Stephenson R.C. Clarke S. J. Biol. Chem. 1989; 264: 6164-6170Abstract Full Text PDF PubMed Google Scholar, 36Wright H.T. Protein Eng. 1991; 4: 283-294Crossref PubMed Scopus (137) Google Scholar) because Gly has more freedom in its main chain torsion angles. This effect was measured by altering Asn-Gly sequences in lysozyme; substitution of Ala for Gly in these sequences protected the enzyme against irreversible heat inactivation (37Tomizawa H. Yamada H. Hashimoto Y. Imoto T. Protein Eng. 1995; 8: 1023-1028Crossref PubMed Scopus (20) Google Scholar). In human and yeast TIMs, two deamidation-sensitive regions have been identified, one in loop 1 and one in loop 3 (29Yuan P.M. Talent J.M. Gracy R.W. Mech. Ageing Dev. 1981; 17: 151-162Crossref PubMed Scopus (70) Google Scholar, 38Gracy R.W. Lu H.S. Yuan P.M. Talent J.M. Adelman R.C. Roth G.S. Altered Proteins and Ageing. CRC Press, Inc., Boca Raton, FL1983: 27-34Google Scholar, 39Casal J.I. Ahern T.J. Davenport R.C. Petsko G.A. Klibanov A.M. Biochemistry. 1987; 26: 1258-1264Crossref PubMed Scopus (60) Google Scholar). Considering all of these factors, it has been proposed that the N12H and G13K replacements in bTIM could be deamidation-preventing molecular adaptations of the enzyme (17Delboni L.F. Mande S.C. Rentier-Delrue F. Mainfroid V. Turley S. Vellieux M.D. Martial J.A. Hol W.G.J. Protein Sci. 1995; 4: 2594-2604Crossref PubMed Scopus (114) Google Scholar). Since the same replacements are also observed in tTIM, it was interesting to see whether this might be part of a general thermostabilization process in TIM. In the present work we have produced double mutants by reintroducing Asn and Gly residues at positions 12 and 13, respectively, in thermophilic bTIM and tTIM. We have measured the activity and structural stability of the mutant enzymes and resolved the three-dimensional structure of the bTIM double mutant. We discuss the individual role of each residue in bTIM and tTIM stability. The E. colistrain HB101 (SupE44 hsd S20 rB-mB recA13ara-14 pro A2 lacY1 gal K2rpsL20xyl-5mtl-l) was used for DNA cloning. The E. coli strain BL21(DE3) (hsd S gal cIts 857 ind1 Sam7 nin5 lacUV-T7, gene 1) was used for protein production. The tTIM gene was overexpressed in the pARAE vector (40L'Hoir C. Renard A. Martial J.A. Gene (Amst.). 1990; 89: 47-52Crossref PubMed Scopus (41) Google Scholar), a derivative of pAR3040 (41Rosenberg A.H. Lade B.N. Chui D.S. Lin S.W. Dunn J.J. Studier F.W. Gene (Amst.). 1987; 56: 125-135Crossref PubMed Scopus (1044) Google Scholar). T. maritima genomic DNA was kindly provided by N. Glansdorff (Research Institute of the CERIA-COOVI, Brussels, Belgium). A polymerase chain reaction (PCR) was performed on T. maritima genomic DNA, using degenerated primers corresponding to conserved regions of TIM (5′-GGNAAYTGGAA-3′ and 5′-NGTNCCNATNGCCCA-3′) (22Rentier-Delrue F. Mande S.C. Moyens S. Terpstra P. Mainfroid V. Goraj K. Hol W.G.J. Martial J.A. J. Mol. Biol. 1993; 229: 85-93Crossref PubMed Scopus (75) Google Scholar). A 500-base pair fragment of the TIM gene was obtained. The entire TIM gene sequence was obtained by inverse PCR (42Ochman H. Medhora M.M. Garza D. Hartl D.L. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego1990: 219-227Google Scholar). Briefly, T. maritima genomic DNA was cut withEcoRI, and fragments were recircularized by ligation. The ligated fragments were used as templates for PCR, using primers complementary to sequences located near the ends of the 500-base pair TIM fragment. The amplification reaction resulted in a head-to-tail arrangement of the sequences originally flanking the target region. This product was cloned and sequenced. To obtain the complete TIM gene, we performed another PCR starting with T. maritimachromosomal DNA, using primers corresponding to regions flanking the TIM gene. The tTIM gene PCR product was cloned into the pCRII plasmid (TA cloning system, Invitrogen Corp.). Several clones were sequenced in both directions in order to find clones without errors potentially introduced by Taq polymerase. The correct clone was called gTm10. The tTIM gene was isolated from the gTm10 vector by PCR, using the following primers: 5′-GCTCTAGAGGCATATGATAACTCGTAAACTG-3′, containing XbaI and NdeI restriction sites (underlined), followed by the first five codons encoding the first five amino acid residues of tTIM, and 5′-TGTTTCTCCTTCGTTTCTCT-3′, complementary to the tTIM nucleotide sequence located 466–485 base pairs downstream from the ATG. The PCR product was cut withXbaI and AvaI, purified and ligated to the digested gTm10 plasmid, and finally sequenced. This new plasmid was then cut with NdeI and BamHI to isolate the entire TIM gene, which was subsequently ligated into theNdeI and BamHI sites of the expression vector pARAE. The plasmid obtained was called pT7-Ther. The expression vector used for bTIM production (named pT7im-Bac) was from Rentier-Delrue et al.(22Rentier-Delrue F. Mande S.C. Moyens S. Terpstra P. Mainfroid V. Goraj K. Hol W.G.J. Martial J.A. J. Mol. Biol. 1993; 229: 85-93Crossref PubMed Scopus (75) Google Scholar). Site-directed mutagenesis was performed on the pT7-Ther and pT7im-Bac vectors using the Chameleon Kit (Stratagene). Double mutants of tTIM and bTIM, containing the H12N and K13G replacements, were constructed using the following oligonucleotides: 5′-CCGAGATCGTGCCGTTCATCTTCCAGTTCC-3′ (for tTIM) and 5′-CCGCTAATGTGCCGTTCATTTTCCAGTTGCC-3′ (for bTIM) (mutated codons are underlined). The individual mutations produced in the bTIM gene were incorporated using the following oligonucleotides: 5′-CCGCTAATGTTTTGTTCATTTTCCAGTTGCCTGC-3′ (for the H12N replacement) and 5′-CCGCTAATGTGCCATGCATTTTCCAGTTGCCTGC-3′ (for the K13G replacement). The oligonucleotides were purchased from Eurogentec, S. A. (Seraing, Belgium). The proteins were overproduced using the T7 system (43Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4854) Google Scholar).E. coli BL21(DE3) cells carrying the expression vectors were grown for 16 h at 37 °C in L broth medium containing 100 mg/l ampicillin. Induction with isopropyl-β-d-thiogalactopyranoside was unnecessary for protein production. Under these conditions, the proteins were produced in a soluble form. The cultures were centrifuged for 15 min at 4500 × g, and the pellets were resuspended in 20 mm triethanolamine HCl buffer, pH 7.6. The cells were disrupted in a high pressure cell (Inceltech, S. A.), and the cell debris was eliminated by centrifugation (45 min, 10,000 ×g). The supernatants were fractionated by ammonium sulfate precipitation, and the TIM-containing fractions were dialyzed overnight at room temperature against 20 mm triethanolamine HCl buffer, pH 7.6. The dialyzed samples were then applied to a Mono Q ion exchange column (HR 10/10, Amersham Pharmacia Biotech) pre-equilibrated in the same buffer. Proteins were eluted from the column with a 0–500 mm NaCl gradient, and the TIM-containing fractions were pooled. Samples containing wild-type TIM were incubated for 10 min at 70 °C and centrifuged for 30 min at 10,000 × g. Sample purity was monitored by SDS-polyacrylamide gel electrophoresis (15% polyacrylamide). Protein concentrations were determined by the Bio-Rad protein assay with bovine serum albumin as the standard. TIM activity assays were performed as described by Misset and Opperdoes (44Misset O. Opperdoes F. Eur. J. Biochem. 1984; 144: 475-483Crossref PubMed Scopus (103) Google Scholar). The assay mixture contained 0.24 mm NADH (Roche Molecular Biochemicals), 20 μg/ml glycerol-3-phosphate dehydrogenase (Roche Molecular Biochemicals), 9.75 mmd-glyceraldehyde 3-phosphate (Sigma), and 100 mm triethanolamine HCl buffer (pH 7.6). The assay was started by the addition of the following enzyme: 0.8 ng of tTIM, 8.9 ng of bTIM, 4.2 ng of the H12N/K13G bTIM double mutant, 32 ng of the H12N/K13G tTIM double mutant, 11 ng of the H12N bTIM mutant, or 7.4 ng of the K13G bTIM mutant. The thermal stability of each protein was tested by incubating the reaction mixtures at temperatures ranging from 25 to 95 °C in a Trio-thermoblock TB-1 (Biometra BAU). Residual activity after incubation was measured at 25 °C. The inactivation rate constant at each temperature (k inact, expressed in s−1) is defined as the slope of the ln(A t/A o) versus the incubation time (22Rentier-Delrue F. Mande S.C. Moyens S. Terpstra P. Mainfroid V. Goraj K. Hol W.G.J. Martial J.A. J. Mol. Biol. 1993; 229: 85-93Crossref PubMed Scopus (75) Google Scholar). Differential scanning calorimetry was performed in a Micro Calorimetry System differential scanning calorimetric unit (MicroCal, Inc.). The protein concentration was adjusted to 1.5 mg/ml for all proteins. Scanning was from 25 to 110 °C (tTIM and H12N/K13G tTIM) and from 25 to 90 °C (bTIM and its mutants) at a scan rate of 1 °C/min. Crystals were grown at 20 °C using the hanging drop vapor diffusion technique under conditions similar to those used to crystallize the wild-type enzyme. The protein solution contained 7 mg/ml protein in 5 mm MES (pH 6.5), 1 mm EDTA, 1 mm sodium azide, 2 mm 2-phosphoglycolate (an inhibitor of the protein). Drops were prepared by mixing 4 μl of protein solution with 3 μl of reservoir solution composed of 25% (w/v) polyethylene glycol 4000, 8% isopropyl alcohol, 100 mm acetate buffer (pH 5.0), 1 mm EDTA, 1 mm sodium azide, and 2 mm dithiothreitol. Needle-shaped crystals were formed. Data were collected to 2.4 Å from one crystal at 20 °C on a Big Mar image plate at station 9.5 of the Daresbury synchrotron source. A wavelength of 1.1 Å was used. The data set was processed with DENZO (45Otwinowski Z. DENZO: An Oscillation Data Processing Program for Macromolecule Crystallography. Yale University, New Haven, CT1993Google Scholar). Scaling, merging, and reduction of the integrated intensities were done with SCALEPACK (45Otwinowski Z. DENZO: An Oscillation Data Processing Program for Macromolecule Crystallography. Yale University, New Haven, CT1993Google Scholar) and TRUNCATE (46Collaborative Computational Project 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19823) Google Scholar). The crystal lattice is primitive orthorhombic with a = 78.13 Å,b = 107.91 Å, and c = 70.98 Å. Because the cell parameters were the same as for native bTIM, the same packing as in the native cell was expected, belonging to space group P21212. Hence, no molecular replacement was done. The coordinates of native bTIM (1BTM; Protein Data Bank, Brookhaven National Laboratory, Upton, NY) were immediately used for rigid body refinement. The R-factor for data between 9 and 3.0 Å dropped to 26.6% for this solution. Data collection statistics are summarized in Table I.Table ICrystallographic data and refinement statisticsParametersValuesCrystal dataSpace groupP21212Cell dimensions (Å)78.132; 107.913; 70.980Cell dimensions (degrees)90.0 90.0 90.0Subunits per asymmetric unit2Data collection statisticsObserved reflections70,035Unique reflections22,732Overall range (Å)30.0–2.4Overall R-merge (%)7.3Overall completeness (%)94.3Last shell range (Å)2.49–2.40Last shell R-merge (%)27.8Last shell completeness (%)65RefinementProtein atoms3672Ligand atoms18Solvent atoms121Resolution range (Å)30.0–2.4R-factor (%)17.5R-free (%)22.0r.m.s. bond length deviations (Å)0.006r.m.s. bond angle deviations (degrees)1.253r.m.s. ΔB for bonded main chain atoms (Å2)1.392r.m.s. ΔB for bonded side chain atoms (Å2)2.529AverageB-factor, all protein atoms (Å2)27.9Average B-factor, backbone atoms (Å2)27.1Average B-factor, side chain atoms (Å2)28.8Average B-factor, ligand atoms (Å2)25.7Average B-factor, solvent atoms (Å2)29.7Ramachandran plotaAs defined by PROCHECK (50).Most favored regions (%)92.3Additionally allowed regions (%)7.7Generously allowed regions (%)0.0Disallowed regions (%)0.0a As defined by PROCHECK (50Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 24: 946-950Google Scholar). Open table in a new tab A few rounds of visual inspection and improvement followed by computer refinement took place. On the one hand, the model was optimized with the program O (47Jones T.A. Zou J.Y. Cowan S.W. Kjelgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13016) Google Scholar) running on an SGI workstation, to improve its fit into a 2Fo-Fc density map. On the other hand, refinement was pursued by combining simulated annealing x-ray refinement and conventional positional and thermal factor refinement, using the X-PLOR (48Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar) package. For refinement, a subset of 5% of the data (the test set) was used for R-free calculations. A bulk solvent correction was applied. In one of the first rounds, the sequence was changed to the mutated one in both monomers. Electron density maps indicated a closed conformation for loop 6, which was manually rebuilt into the density map. In addition and as expected, the 2-phosphoglycolate molecule was found in the active site of each subunit. Its coordinates were included in the model. The final refinement stages were carried out with crystallography and NMR system (49Brünger A.T. Adams P.D. Clore M.G. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16989) Google Scholar). Water molecules were added at sites displaying a peak larger than three S.D. values above the mean in an Fo-Fc map and having a potential hydrogen bonding partner. A total of 121 water molecules were identified. The final R-factor is 17.5%, and R-free is 22.0% for all data in the resolution range from 30 to 2.40 Å. The quality of the structure was analyzed with the programs PROCHECK (50Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 24: 946-950Google Scholar) and WHAT IF (51Vriend G. J. Mol. Graphics. 1990; 8: 52-56Crossref PubMed Scopus (3379) Google Scholar). The refinement statistics are summarized in TableI. Energy minimizations (molecular dynamics and molecular mechanics) were performed with the Discover program DM1 (Discover User Guide, Molecular Simulations, San Diego), using the cvff force field. Graphical displays were generated with the InsightII molecular modeling system DM2 (InsightII User Guide, Molecular Simulations, San Diego). Computations were done on a Silicon Graphics Indigo2 workstation running Irix 5.3. The three-dimensional coordinates of bTIM, tTIM, and H12N/K13G bTIM were taken from the corresponding crystallographic structures for bTIM (1BTM; Protein Data Bank, Brookhaven National Laboratory, Upton, NY), tTIM,2and the double mutant bTIM (this work). Three-dimensional structures of the single mutants H12N bTIM and K13G bTIM were generated with the Builder interface of the InsightII program, starting with the experimental structure of bTIM. The coordinates of the ligand (2-phosphoglycolate in bTIM and a sulfate ion in tTIM) were retained in the structures, while water molecules were removed. Hydrogens were automatically assigned. A distance-dependent (1.r) dielectric constant was used. For simulations, a subset was defined for each structure, containing all amino acids of the protein within 18 Å of the active site. All coordinates (main chain and lateral chain) were fixed except those of loop 1 (residues 9–20 and 11–22 in bTIM and tTIM, respectively). A combination of molecular mechanic runs (steepest descent + conjugated gradient + Newton-Raphson) was first applied to the structures in order to define equivalent inputs for the molecular dynamics simulations. This was done as the starting structures were obtained at different levels of precision (different resolutions). Molecular dynamics ensued at 300 K for a total of 100 ps (20 runs of 5000 fs), generating 20 geometries for each structure. The geometries were further minimized (annealing to a final derivative cut-off of 0.05 kcal/mol) and analyzed. By in vitro site-directed mutagenesis we replaced the residues His12 and Lys13 of bTIM and tTIM with Asn and Gly, respectively, these two residues being often found in mesophilic TIMs. The two single poi

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