Báo cáo khoa học: Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human aB-crystallin docx

Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human aB-crystallin Melissa M. Valdez 1 , John I. Clark 1,2 , Gabrielle J. S. Wu 3 and Paul J. Muchowski 4 1 Departments of Biological Structure, and 2 Ophthalmology, University of Washington, Seattle, WA, USA; 3 Seattle Genetics, Bothell, WA, USA; 4 Department of Pharmacology, University of Washington, Seattle, WA, USA Mycobacterium tuberculosis heat shock protein 16.3 (MTB HSP 16.3) accumulates as the dominant protein in the latent stationary phase of tuberculosis infection. MTB HSP 16.3 displays several characteristics of small heat shock proteins (sHsps): its expression is increased in response to stress, it protects against protein a ggregation in v itro, and it contains the core Ôa-crystallinÕ domain found in all sHsps. In this study we characterized the c haperone activity of r ecombinant MTB HSP 16.3 in several different assays and compared the results to those obtained with recombinant human aB-crystallin, a well characterized member of the sHsp family. Recombinant MTB HSP 16.3 w as expressed in Escherich ia coli and purified to apparent homogeneity. Similar to aB-crystallin, MTB HSP16.3 suppressed citrate synthase aggregation and in the presence of 3.5 m M ATP the chaperone activity was e nhanced by twofold. ATP stabilized MTB HSP 16.3 against proteolysis by chymotrypsin, and no effect was o bserved w ith ATP cS, a nonhydrolyzable analog of ATP. Increased expression of MTB H SP 16.3 resulted in protection against thermal killing in E. co li at 48 °C. While the sequence similarity between human aB-crystallin and MTB HSP 16.3 is only 18%, these results suggest that the functional similarities between t hese proteins containing the core Ôa-crystallinÕ domain are much closer. Keywords: ATP; human aB-crystallin; molecular chaperone; Mycobacterium tuberculosis HSP 16.3 ; small heat s hock proteins. One-third of the world’s population is infected with latent inactive tuberculosis and active tuberculosis is the leading cause of d eath due to an infectious disease [ 1]. Each year, new infections occur in 54 million people; 6.8 million people develop c linical disease, and 2 .4 million cases result in death [2]. There is still limited knowledge of the molecular pathogenesis of the latent stage of this organism [3]. Individuals w ho have been infected with Mycobacterium tuberculosis can harbor stable dormant bacilli for decades before developing an active infection later in life [4]. Recent reports indicate an important role for M. tuberculosis (MTB) heat shock protein (HSP) 16.3 in the survival of MTB during p rolonged periods of infection [5–7]. It w as shown that MTB HSP 16.3, initially described as the immunodominant 14- or 16-kDa antigen [8–11], was a major component in tuberculosis infection in humans and played an important role in enhancing protein stability and survival [5]. Eighty-five percent of patients with active tuberculosis showed a positive reaction to this a ntigen, suggesting that this protein expressed in vivo had a key role in MTB infection [11,12]. The 14K antigen was later renamed MTB HSP 16.3 [13]. MTB HSP 1 6.3 accumulates to become t he dominant protein in the la tent stationary phase of M. tube rculosis infection [7]. Over-expression of HSP 16.3 in log phase growth of M. tuberculosis slowed the growth rate and protected ag ainst stationary phase autolysis in v itro [7]. MTB HSP 16.3 h as been charac terized a s a membrane associated protein [12] having sequence homo- logy to other proteins i n the small heat s hock protein (sHsp) family [11,14]. All sHsps share se quence similarity in a conserved 80–100 amino-acid Ôa-crystallinÕ domain region found in the C-terminus which is thought to be important for c haperone function s [14–16]. MTB HSP 16.3 h as been shown to contain an oligomeric, active structure which may form a trimer of trimers and pos sesses in vitro molecular chaperone activity [13]. Up-regulation o f l arge and small sHsps is t hought to be a universal response to s tress. In vitro, human aB-crystallin and other sHsps function as molecular chaperones by suppressing unfolding and aggregation o f polypeptides in response to s tress [17,18]. MTB HSP 16.3 modulates its chaperone activity by exposing hydrophobic s urfaces a nd demonstrates conformational flexibility allowing maximum interactions with denaturing proteins [19]. A recent paper reports that th e only universally con served leucine residue among all the members of the sHsp family plays an important role i n m olecular c haperone activity of MTB HSP 16.3 and oligomeric structure formation [20]. I t has been reported t hat the chaperone activity of MTB H SP 16.3 is independent of the effects of ATP [13,19]. In contrast, molecular chaperones of the large heat shock protein families suppress protein unfolding and aggregation during stress and participate in the refolding of denatured proteins in vitro, in an ATP-dependent manner [21,22]. While the molecular chaperone effects of the sHsp do not r equire ATP, the activity of human- aB crystallin was enhanced with Correspondence to J. I. Clark, Department of Biological Structure, Box 357420, University of Washingto n, Seattle, W A 98195-7420, USA. Fa x: +1 206 543 1524, Tel.: 1 206 685 0950, E-mail: clarkji@u.washington.edu Abbreviations: MTB HSP 16.3, Mycobacte rium Tuberculosis heat shock protein 16.3; sHsps, small heat shock proteins; IPTG, isopropyl thio-b- D -galactoside; CFUs, colony forming units; CS, citrate synthase. (Received 12 July 2001, r evised 1 7 January 2 002, accepted 25 January 2002) Eur. J. Biochem. 269, 1806–1813 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02812.x ATP [23]. Recent reports confirmed the effect of ATP on sHsps using bovine a-crystallin to stabilize partially dena- turedproteinsduringreactivationinanATP-dependent manner [24] and indicating the involvement of ATP in substrate release [25]. In this s tudy, t he effect of ATP on recombinant MTB HSP 16.3 was compared with the effect of ATP on recombinant human aB-crystallin. We report t he expression and purification of recombinant MTB HSP 16.3 from E. coli and studies of its f unction as a molecular chaperone. MTB HSP 16.3 was compared with aB-crystallin in vivo and in vitro biochemically and in functional assays. Although only 18% sequence identity is shared between the two s Hsps, MTB HSP 16.3 functioned as effectively as aB-crystallin as a molecular chaperone in v itro. The molecular chaperone activity of recombinant MTB HSP 1 6.3 was enh anced in the presence of ATP which is consistent with previous findings of the effect of ATP on recombinant human aB-crystallin [23]. The expression of MTB HSP 16.3 in E. coli exposed to high temperatures resulted in a very impressive level of survival. Our results suggest the chaperone activity ofMTB H SP 16.3may play an important role in the survival and stability of M. tuberculo sis. MATERIALS AND METHODS Expression and purification of MTB HSP 16.3 HSP 1 6.3 was subcloned into the pET-20b(+) expression vector which w as provided by H. McHaourab (Department of Molecular Physiology a nd Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA). T he pET-20b(+)-HSP 16.3 expression plasmid was used to transform E. coli BL21 (DE3) competent cells (Novagen, Inc., Milwaukee, WI, USA). The expression of HSP 16.3 was based on a method described previously [26]. One litre of Luria–Bertani broth containing 10 g NaCl, 5 g yeast extract, and 10 g tryptone (DIFCO Laboratories), p H 7.0 with 50 lgÆmL )1 carbenicillian was inoculated with 10 mL of an overnight c ulture containing the pET-20b(+)- HSP16.3 vector. The flasks were incubated for a total of 3 h at 37 °C until D 600 ¼ 0.8–1.0. The cells were then induced with 0.4 m M isopropyl thio-b- D -galactoside (IPTG) for another 4 h. Cells were then harvested by sedimentation and frozen at )20 °C until further use. Cell pellets of the pET-20b(+)-HSP 16.3 were then lysed with 1 0 mL l ysis buffer (20 m M Tris/HCl pH 7.0) and transferred to a small beaker which was placed in ice. Forty microliters of 50 m M phenylmethanesulfonyl fluoride and 400 lL10mgÆmL )1 lysozyme were added with constant stirring for 10 min; 20 mg deoxycholic acid were then added with an additional 10 min of stirring. The m ixture was removed from the ice bath and 200 lLof1mgÆmL )1 DNAse was added with stirring for 30 min. The sample was then placed in a 50-mL tube and centrifuged at 18 000 g for 1 h. The supernatant from this sample was transferred to a new beaker with constant stirring at room temperature with the addition of 400 lL 5% polyethylenimine a nd 800 lL 200 m M dithio- threitol for 10 min. The sample was then c e ntrifuged at 35 000 g for 2 h at 4 °C. The supernatant was decanted and the insoluble pellet w as discarded. The s upernatant was then ready for purification using the Pharmacia FPLC system. The supernatant containing the soluble protein was filtered through a 0 .22 lm filter and was loaded onto a High Trap Q Anion Exchange Column (Pharmacia), pre-equil- ibrated with Buffer A (20 m M Bis/Tris, pH 6.5). The proteins were eluted using a linear gradient of 0–1.0 M NaCl. T he protein fractions were analyzed using SDS/PAGE (Invitrogen). Proteins were analyzed on a 4–12% polyacrylamide e lectrophoretic gel in the presence of 0.1% SDS a nd Mes buffer and were stained with C oomasie blue R-350 (Amersham Pharmacia). Fractions containing the 16.3-kDa p rotein were th en pooled and concentrated using a 1 0 000 molecular mass cut-off concentrator (Amicon). Concentrated protein (5 mL) was loaded onto a Phenyl Superose Hydrophobic Interaction Column, preequilibrated with a buffer containing 50 m M sodium phosphate, and 1.0 M ammonium sulfate, pH 7.0. The protein was then eluted using 50 m M sodium phosphate, pH 7.0. The protein fractions were analyzed by SDS/PAGE and all fractions containing the protein were pooled and concentrated to 500 lL. The concentrated sample was applied to a Superdex 200 HR 10/30 size exclusion column (Pharmacia) and purified by gel filtration. Fractions containing the p rotein w ere collected, c oncentrated, a nd then q uantified for total protein concentration u sing the Bradford method (Bio-Rad). To confirm protein purification of HSP 16.3, a Western immunoblot analysis was performed using monoclonal antibody IT-4 (a-16 kDa) provided by D. Sherman (Department of Pathobiology, University of Washington, Seattle, WA, USA). Detection of protein was perfo rmed using NEN Western Blot Chemiluminescence Reagents (NEN Life Science P roducts Inc.). The s eq uence of t he purified recombinant HSP 16.3 was confirmed b y MS. Automatic Edman sequencing was used with an applied Biosystems model 470 A automatic protein sequencer. In vivo cell viability experiment with HSP 16.3 at 48 °C A XbaI–XhoI fragment containing the entire coding sequence of HSP 16.3 was excised from pET20b(+)- HSP 1 6.3 and ligated into pET16b (Pharmacia) digested with the same restriction enzymes. Restriction mapping analysis was performed on pET16b-HSP 16.3 to ensure that the HSP 16.3 gene was i nserted in the proper orientation, and the coding sequence of HSP 16.3 was verified by DNA sequence analysis. The pET16b-HSP 16.3 vector was then t ransformed into BL21 (DE3) competent cells. For t he thermal killing experiment, an equal number of cells were gr own containing either pET16b-HSP 16.3 vector, pET16b (empty vector control), or pET16b-aB (positive control). Equal numbers of cells from overnight cultures were inoculated into 50 mL of L -broth medium containing 100 lgÆlL )1 carbenicillin and grown at 37 °C until they reached an D 600 ¼ 0.8. Protein expression was then induced with 1.0 m M IPTG. After a 2 h i nduction, samples were s hifted to a s haking water bath a t 4 8 °C. Samples w ere r emoved at 3-h t ime points postinduction and scored f or cell viability by p lating on Luria–Bertani broth plates containing carbenicillin. Cell viability was determined by counting the number of colony forming units (CFUs) on each plate after heat shock at 48 °C relative to the s tarting number of CFUs f ormed in each culture p rior to heat shock. Total protein lysates from cells that expressed HSP 16.3 and control cultures w ere Ó FEBS 2002 Similarities between MTB HSP 16.3 and aB-crystallin (Eur. J. Biochem. 269) 1807 analyzed by SDS /PAGE as described above. The experi- ment was repeated four times using duplicates of each cell culture. Chaperone assays The t hermal unfolding and aggregation of citrate synthase (CS; Roche Molecular Biochemicals) at 4 5 °Cwasdeter- mined by measuring the absorption from light scattering at 320 n m in a Beckman Spectrophotometer over a period of 30 min. Native CS was diluted to a 15 l M working solution containing 20 m M Tris/HCl (pH 7 .4), and 100 m M NaCl. To t est the molecular chaperone effect, different molar ratios of HSP 16.3 were diluted into a reaction buffer containing 100 m M Tris/HCl (pH 7.4), 100 m M NaCl in the presence or absence of ATP (final volume 400 lL). For testing the effects of A TP and the nonhydrolyzable ATP analog ATPcS, the r eaction buffer was equilibrated w ith 3.5 m M ATP (or A TPcS), 3 .5 m M MgCl 2 and 1 0 m M KCl b efore addition of HSP 16.3 o r CS. The protection from thermal aggregation of CS at 45 °C w ith HSP 16.3 was a lso c ompared with t he same molar ratios of aB-crystallin (predicted from monomeric molecular weights). Chymotrypsin digestion of HSP 16.3 Chymotrypsin digestion with HSP 16.3 was base d on the methods used for GroEL and aB-crystallin [27,28]. In summary, f or each reaction 70 lgMTBHSP16.3were diluted into a final volume of 100 lL buffer c ontaining 100 m M Tris/HCl, pH 7.4, 3.5 m M MgCl 2 ,10m M KCl, and 0.01% Tween-20. For reactions with ATP or ATPcS, a final concentration of 3.5 m M ATP or ATPcSwas added to th e reaction mixture. To each sample at time point 0, 0.17, 0.51 or 1.36 lg chymotrypsin was added from a stock solution of 0.17 mgÆmL )1 .Sampleswere maintained at 37 °C f or the duration o f the experiment. Immediately after chymotrypsin addition, 13.5 lLofthe reaction mixture was removed and quenched with 1.5 lL 100 m M phenylmethanesulfonyl fluoride, and placed on ice. At 5-min time points, 13.5-lL aliquots were r emoved and treated identically to the zero time point sample. Samples were analyzed by SDS/PAGE analysis as described above. RESULTS Expression and purification of recombinant MTB HSP 16.3 in E. coli Figure 1 is the SDS/PAGE and Western immunoblot analyses of the expression and purification of MTB HSP 16.3. Induction of protein expression with IPTG resulted in the a ppearance of a protein band at approximately 16.3 k Da (Fig. 1 A, lanes 2 and 3). The expressed M TB HSP 16.3 was purified by a combination of anion e xchange, hydrophobic i nteraction and s ize exclusion chromatography (lanes 4–6). Wester n immuno- blot analysis of recombinant MTB HSP 16.3 was performed using a monoclonal antibody [IT-4 (a-16 kDa)] raised against native MTB HSP 1 6.3 (Fig. 1 B). IT-4 (a-16 kDa) recognized the recombinant MTB HSP 16.3 from E. coli and did not react with recombinant aB-crystallin (Fig. 1 B). The purification yield obtained from 1 L of Luria–Bertani broth culture was between 10 and 3 0 mg o f MTB HSP 16.3. Fig. 1. Expression and Purification of MTB HSP 16.3. (A) The expression an d p urification of MTB HSP 16.3 was a nalyz ed b y S DS/ PAGE using 4–12 % Bis/Tris polyacrylamide gels in the presence of Mes buffer. Lanes 1 and 7, m olec ular mass markers; lane 2, expression protein in E. coli cells not induced by IPTG; lane 3, protein expression in E. coli after induction with IPTG; lane 4, following purification on the High trap Q anion exchange c olumn; lane 5, MTB HSP 16.3 enriched after purification using a Phenyl Superose H ydro phobic Interaction Column; lane 6, following Tris/HCl buffer exchange of MTB HSP 16.3 on a Superdex 200 Size Exclusion column (to remove salt). A protein assay performed after desalting the sample showed that the y ields varied between 10 and 30 mgÆL )1 of MTB HSP 16.3 cell culture. (B) SDS/PAGE (left) and Western immunoblot (right) on a 4–12% polyacrylamide gel of recombinant MTB HSP 16.3 and recombinant h uman aB-crystallin. Lane 1, molecular mass markers; lane 2, MTB HSP 16.3; lane 3, aB-crystallin. In the Western immu- noblot, th e I T-4 antibody to MTB HSP 16.3 d etected r ecom binant MTB HSP 16.3 in lane 2 only. No reactivity with anti-(MTB HSP 16.3) I g was obser ved in lan e 3, which contained human aB-crystallin (right s ide). 1808 M. M. Valdez et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Amino-acid sequence determination of recombinant MTB HSP 16.3 The s equence of the purified recombinant MTB HSP 16.3 was c onfirmed b y M S. T he 1 2 r esidues a t the N-terminus of MTB HSP 16.3 were determined to b e: Ala-Thr-Thr-Leu- Pro-Val-Gly-Arg-His-Pro-Arg-Ser. The N-terminal Met residue was not identified by p rotein sequencing, as observed previously [12,13]. Protection of cell viability at 48 °Cin E. coli by MTB HSP 16.3 and aB-crystallin To characterize the protective e ffect of MTB HSP 16.3 over-exp ression in vivo, a thermal k illing assay in E. coli was used (Fig. 2A). The number of CFUs w ere counted in cultures of E. coli with and without MTB H SP 16.3 and aB-crystallin shifted to 48 °C. The proportion of viable cells that survived heat shock at 48 °C was plotted at three time points (t ¼ 0, 3 and 6 h a fter 48 °C heat shock). C ells that expressed MTB HSP 16.3 or aB-crystallin were resistant to thermal killing a t 48 °C over t he 6-h time c ourse of the experiment (Fig. 2A). After 3 hs, the viability of cells th at contained the empty c ontrol vector decreased b y four orders of magnitude, while the viability of cells that over-expressed MTB HSP 16.3 o r aB-crystallin decreased only slightly. Within 6 h of heat shock a t 48 °C, no viable ce lls were observed in cells containing the aB-crystallin vector that were not induced for protein e xpression. The viability by 6 h , decreased dramatically in cells containing the pET 16b control vector as measured by the CFUs. In contrast, the viability o f cells that over-expressed MTB HSP 16.3 decreased by two orders of magnitude after the 6-h heat shock. The protective effect of the induced aB-crystallin was stronger than MTB HSP 16.3. SDS/PAGE analysis s howed strong expression of MTB HSP 16.3 and aB-crystallin at time points 0 , 3, and 6 h following heat shock at 48 °C (Fig. 2 B,C). In vitro chaperone activity of MTB HSP 16.3 and aB-crystallin In Fig. 3, we observed the effects of different concentrations of MTB HSP 16.3 in the presence and absence of ATP on the aggregation of CS. In the absence of added MTB HSP 1 6.3, aggregation o f CS increased af ter a short delay to reach a maximum after approximately 25 min at 4 5 °C Fig. 2. Cell viability of MTB HSP 16.3. The pET 16b-MTB HSP 16.3 vector, the control pET 16b-aB vector and the pET 16b vector con- taining no inserted gene were e xpressed at 37 °C and induced with IPTG when the cell c ultures reached D 600 ¼ 0.8. After induction fo r 2 h and heat shock to 48 °C, the cells were incubated for a further 6 h. Samples were taken at concurrent and sequential time points beginning at the time of heat shock, plated and CFU counted. The proportions of viable cells expressing the pET 16b-MTB HSP 16.3 vector and the two control vectors were plotted for 0 , 3 and 6 h following h eat s hock. At 48 °C, the proportion of surviving cells expressing pET 16b vector only or aB-crystallin uninduced c ells was negligible and viability of cell cultures decreased more than fourfold (A). By 6 h post heat shock, the cultures that over-expressed MTB HSP 16.3 and the aB-crystallin in- duced c ells remained viable. Protein expression was analyzed b y SDS/ PAGE on a 4–12% polyacrylamide gel in the presence of 0.1% SDS and Mes bu ffer (B,C). Lanes 1 a nd 6 are molecular mass markers. Lanes 2–5 show protein expression i n cells containing the pET 16b vector alo ne a t selected times f rom 0 to 6 h. Lane 2 is the pro tein expression in cells containing the pET 16b vector alone not induced with IPTG. Lanes 3–5 are the pET 16b vector at the zero, 3 and 6 h time points after 2 h of induction and post heat shock at 48 °C. Lane 7 of (B) i s protein ex pression in cells containing t he pet 16b-MTB HSP 16.3 vector not in duce d with IPTG. Lanes 8 –10 a re pro tein expression for the pET 16b-MTB HSP 16.3 vector at time point zero, 3 and 6 h after 2 h of induction and post heat shock. (C) SDS/PAGE of cells containing aB-crystallin induced and not induced with IPTG. Lane 2–5 show aB-cr ystallin uninduced at the 0, 3 and 6 h time points. Lane 7 contains cell homogenates of aB-crystallin n ot induced. Lane s 8–10 contain aB-crystallin induced with IPT G at the 0 , 3 and 6 h time points. The cells expressing high levels of MTB HSP 16.3 or aB- crystallin (lanes 8–10) survived in culture at 48 °C(A). Ó FEBS 2002 Similarities between MTB HSP 16.3 and aB-crystallin (Eur. J. Biochem. 269) 1809 (Fig. 3 A). With increasing ratios of M TB HSP 1 6.3 to C S, a concentration-dependent suppression of aggregation was observed over the 30-min period (Fig. 3 A). Complete protection against aggregation was observed at a ratio of 15 : 1 MTB HSP 16.3:CS (monomer : monomer). The addition of 3.5 m M ATP enhanced the effect of MTB- HSP 16.3 on CS aggregation by approximately twofold (Fig. 3 B). ATPcS, a nonhydrolyzable analog of ATP, did not enhance t he effect of MTB H SP 16.3 on CS aggregation (Fig. 3 C). T he chaperone activity of MTB H SP 16.3 was next compared to aB-crystallin at identical molar ratios (Fig. 4). In ge neral, aB-crystallin was more effective as a molecular chaperone than MTB HSP 16.3 under the conditions of these experiments (Fig. 4). Complete s uppres- sion of CS aggregation by MTB HSP16.3 required a molar ratio of 15 : 1, while aB-crystallin required a molar ratio of 5 : 1 for complete suppression of aggregation. Chymotrypsin proteolysis of MTB HSP 16.3 in the absence and presence of ATP MTB H SP 16.3 was digested with chymotrypsin in the absence and presence of ATP at 42 °C (Fig. 5A–C). Proteolysis of MTB HSP 16.3 increased with chymotrypsin concentration as expected (data not shown). Each individ- ual lane is a sample of MTB HSP 16.3 plus chymotrypsin Fig. 4. Comparison of molecular chaperone activity between recombin- ant MTB HSP 16.3 and recombinan t human aB-crystallin. The molecular chaperone activity of MTB HSP 16.3 o n CS aggregation was compared to the effect of human aB-crystallin on C S aggregation. The aggregation of CS was mea sured in the presence o f d ifferent concentrations of MT B HSP 16.3 o r human aB-crystallin after a 30-min period. The bar graphs measure the a ggregation of C S in arbitrary units vs. t he ratios of MTB HSP 16.3/CS and h uman aB-crystallin/CS. With increased ratios of the molecular chaperone protein to CS, there was increased protection aga inst CS aggregation. Recombinant human aB-crystallin demonstrated better p rotection against CS aggregation than MTB HSP 16.3. At the 15 : 1 molar ratio of MTB HSP 16.3/CS, protection aga inst CS aggregation was almost complete. Similar protection was observed at a ratio of 5 : 1 for human aB-crystallin/CS. Fig. 3. Molecular Chaperone Activity of MTB HSP 16.3. To test the molecular chaperone activity of MTB HSP 16.3, a series of aggrega- tion assays was perform ed using CS with and without ATP over a 30-min period. The aggregation of CS was measured with the addition of different concentrations of MTB HSP 16.3 and in the presence or absence of ATP and ATP analogs. (A) Aggregation o f CS is plotted in arbitrary units against time in the presence of increasing ratios of MTB HSP 16.3 to CS. With the increase of MTB HSP 16.3, there was an increase of protection against t hermal aggregation of CS [d, CS alone; j, HSP 16.3/CS (5 : 1); ,, H SP 16.3/CS (10 : 1); ., HSP 16.3/CS (12 : 1); s, HSP 16.3/CS (15 : 1)]. (B) Aggregation of CS plotted in arbitrary units against time in the absence a nd presence of ATP at two different molar concentrations of MTB HSP 16.3. In the presence of ATP, the molecular chaperone effect of MTB HSP 16.3 was enhanced for aggregation of CS and maximum suppression of aggregation was observed at a molar ratio of 10 : 1 HSP 16.3 : CS [d, CS a lone; ,, HSP 16.3 : CS (5 : 1); j, HSP 16.3 : CS (5 : 1) + ATP; s, HSP16.3:CS (10:1); ., HSP 16.3 : CS (10 : 1) + ATP]. (C) Control for the effe ct of ATP on t he mole cular c haperone ac tivit y of MTB HSP 16.3. When 3.5 m M MgCl 2 and 1 m M KCl were added to a solution containing MTB H SP 16.3 and CS no effect on chaperone activity was o bserved. The results using A TPcSandMgCl 2 with KCl suggest the im portan ce of hydrolysis of ATP for c haperone activity of MTB HSP 16.3. [d,CSalone;,,HSP16.3:CS (10:1)+1m M KCl and 3.5 m M MgCl 2 ; s, HSP 16.3 : CS (10 : 1); .,HSP 16.3 : CS (10 : 1) + 3.5 m M ATPcS; j, HSP 16.3 : CS (10 : 1) + 3.5 m M ATP]. 1810 M. M. Valdez et al. (Eur. J. Biochem. 269) Ó FEBS 2002 taken at 5-min time intervals over a 30-min period. Nearly all intact MTB HSP 16.3 was degraded after 15 m in in the absence of ATP (Fig. 5A). In the presence of 3.5 m M ATP, MTB HSP 16.3 was stabilized against proteolysis, and intact MTB HSP 16.3 could be detected even after 3 0 min of proteolysis (Fig. 5B). The digestion pattern of MTB HSP 1 6.3 was similar in the absence and presence of ATP, where two major proteolytic fragments at M r  8and 13 kDa were observed. The specificity of the effect of ATP on stabilization of MTB HSP 16.3 against p roteolysis was confirmed with nonhydrolyzable ATP analog ATPcS, which had no stabilizing e ffect against proteolysis (Fig. 5C). DISCUSSION Our results demonstrated similar functions for recombinant MTB HSP 16.3 and human aB-crystallin as molecular chaperones, although the sequence identity between MTB HSP 1 6.3 and aB-crystallin is only 18% (Fig. 6). MTB HSP 1 6.3 is a sHsp that contains the conserved c ore Ôa-crystallinÕ domain shared by m embers of the sHsp family [14–16]. M. tuberculosis HSP 16.3 was expressed and puri- fied from E. coli for the comparative characterization on t he molecular chaperone activity in vivo and in vitro with human aB-crystallin, the well characterized archetype of the sHsp family of molecular chaperones [14,16,29]. In vivo, the protective e ffect of MTB HSP 16.3 expression on the survival of E. coli in a thermal killing assay at 48 °C was impressive (Fig. 2A). At 48 °C, there was approxi- mately two orders of magnitude difference between survi- ving cells expressing MTB HSP 16.3 and c ontrols without MTB HSP 16.3 expression. The results for MTB HSP 16.3 are consistent with previous reports with other sHsps [23,30,31]. The protective effect of aB crystallin on cell survival was s tronger than MTB HSP 16.3. I n p revious in vivo studies, o ver-expression of HSP 16.3 at the end of log-phase growth in M. tuberculosis resulted in an enhanced resistance to autolysis [ 5]. Our results s howing a protective effect of MTB HSP 16.3 against thermal killing in E. coli are consistent with previous studies on the importance o f MTB HSP 16.3 expression in M. tuberculosis [5–7]. Although the role of MTB H SP 16.3 is not completely understood, these experiments suggest that MTB HSP 16.3 may provide protection against cell death in M. tuberc ulosis. In an in vitro aggregation assay using CS as a target protein, MTB HSP 16.3 was effective as a chaperone, although less effective than aB-crystallin in suppressing CS aggregation. It is possible that additional cofactors found only in M. tuberculosis cytosol could i ncrease the chaper one activity of MTB HSP 16.3. It is also likely t hat the efficiency of MTB HSP 16.3 as a chaperone may b e improved using target proteins that are native to M. tuberculosis. The e ffects of ATP on the c haperone activity of MTB HSP 1 6.3 were similar to aB-crystallin, a sHsp whose Fig. 6. Sequence alignment of recombinant MTB HSP 16.3 and recombinan t hum an aB-crystallin. Amino-acid sequence a lignment between recombinant MTB H SP 16.3 and h uman aB-crystallin was aligned using the MULTALIN MULTIPLE SEQUENCE ALIGNMENT program (PBIL, Franc e) with the h elp of S. Yarfitz (Unive rsity o f Washington Health Science s L ibrary, Seattle, WA, USA). Shading indicates chemically identical and similar a mino-acids residues ( BOXSHADE program from the European Molecular Biology Network). Residues highlighted black indicate amino-acid residues that are chemically identical a nd residu es h igh lighted gray indicate amino-acid residues that are c hemically similar. Between MTB HSP 16.3 and human aB-crystallin there was an 18 % sequence identity and a n overall 30% shared sequence similarity between the tw o proteins. The conserved core a-crystallin domain observed in proteins of the sHsp spans resi- dues of E67–I161 in the aB- crystallin sequence. Fig. 5. The chymotrypsin proteolysis of MTB HSP 16.3. SDS/PAGE used 4–12% Bis/Tris polyacrylamide gels in the presence of Mes buffer (A–C). Arrows indicate the MTB HSP 16.3 band. Lane 1 of each gel contains the molecular mass markers. Each individual lane is a sample of MTB HSP 16.3 plus 0.51 lg chymotrypsin taken at 5-min intervals over a 30-min period. MTB HSP 16.3 was readily degraded by chymotrypsin and nearly all i ntact protein was degraded by 15 min (A). In the presence of 3.5 m M ATP, MTB HSP 16.3 was stabilized against proteolysis by chymotrypsin and intact MTB HSP 16.3 remained after 30 min of digestion (B). In the presence of the non hydrolyzable analogue of ATP, ATPcS, there was no stabilization of MTB HSP 16.3 against chymotrypsin proteolysis (C), consistent with the effect of ATPcS on the molecular chaperone function of MTB HSP 16.3 reported i n F ig. 3. Ó FEBS 2002 Similarities between MTB HSP 16.3 and aB-crystallin (Eur. J. Biochem. 269) 1811 chaperone function was enhanced by ATP [23]. In separate reports, ATP increased the refolding of xylose reductase by total a-crystallin [34], increased the binding of a-crystallin to lens membranes, and inhibited the chaperone activity of a plant sHsp [35,36]. C onsistent with previous studies using aB-crystallin, the present results demonstrated that ATP enhanced the chaperone effect of MTB HSP 16.3 by twofold in the CS aggregation assay. However, the specific role of ATP in t he chaperone function of sHsps has been controversial [18,23,28,29]. Previous reports indicated that the chaperone activity of MTB H SP 16.3 may be ATP independent [13,19] while structural studies demonstrated an interaction between ATP and total bovine a-crystallin using equilibrium binding studies, intrinsic tryptophan fluorescence and 31 P NMR [23,25,34,35,37]. aB-Crystallin has a lso been reported to d isplay an autok inase activity [38–40]. R ecent r eports suggest that A TP may participate in the release of tar get peptides from aA-crystallin [24,25]. Here, M TB HSP 16.3 chaperone activity was measured in a Tris/HCl buffer system, while previous studies of MTB HSP 16.3 were performed in either a Hepes/HCl or sodium phosphate buffer systems [13,19]. The conditions used in this study were the same as those used successfully to demonstrate the ATP effect on human aB-crystallin [23]. In separate experiments the chymotrypsin proteolytic digestion pattern of MTB H SP 16.3 in the presence and absence of ATP was evaluated. Similar to aB-crystallin [28] and Hsp27 [32], chymotrypsin cleavage sites in MTB HSP 16.3 appeared to be shielded in the presence of ATP. The s imilarity of the chymotrypsin d igestion pattern for MTB HSP 16.3 to previous studies with aB-crystallin and Hsp27 may indicate similar domain structures and assembly properties that are stabilized in the presence of ATP. As with aB-crystallin, ATPcS (a nonhydrolyzable A TP analog) did not enhance the chaperone function of MTB HSP 16.3, and did not protect against i ts proteolysis by c hymotrypsin. Although there is on ly 18% sequen ce identity, the c ore Ôa-crystallinÕ domain in M TB HSP16.3 may have functional significance similar to that of aB-crystallin [28]. MTB HSP 16.3 of M. tuberculosis may be ideally suited for studies of the structure and function of the core Ôa-crystallinÕ domain of sHsps because the quaternary structure is more monodisperse than aB-crystallin and other sHsps, that are known to have highly variable quaternary structures [ 41]. T he crystal s tructure of HSP 16.5 from Methanococcus janaschii demonstrates a mono- mer containing a core domain that consists largely of b sheets [42]. The molecules o f HSP 16.5 form dimers that assemble into a spherical complex of octahedral symmetry, while MTB HSP 16.3 is reported t o consist of a trimer of trimers [13]. Spin labeling o f MTB HSP 16.3 in solution is consistent with a core domain consisting of a twofold symmetric interface b etween subunits that involved two b strands in the core a-crystallin domain interacting in an antiparallel fashion [43]. While previou s mutagenesis studies demonstrated the functional importance of the core Ôa-crystallinÕ domain in s Hsps [28,29,33], the structural basis for the function of the conserved core Ôa-crystallinÕ domain remains to be defined. New strategies are needed to understand the precise mechanism that allows the tubercle bacilli of M. tuberculosis to survive long-term dormancy. This study supports the hypothesis t hat the conserved core Ôa-crystallinÕ domain i n MTB HSP16.3 may be important for long-term dormancy in M. tuberculosis.Futurein vivo studies of specific inter- actions between MTB HSP 16.3 and other latent stage proteins will lead to a b etter u nderstanding of the molecular chaperone activity of MTB HSP 16.3. Further structure– function analyses including the determination of an atomic resolution model of MTB HSP 16.3 are needed. Crystallo- graphic studies of HSP 16.3 could be used to determine sites for interactions with other proteins and/or ATP. 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(1998) Cr ystal structure of a small heat-shock prot ein. Nature 394, 595–599. 43. Berengian, A.R., Parfenova, M. & McHaourab, H.S. (1999) Site- directed spin labeling study of subunit interactions in the alpha- crystallin domain of small heat-shock proteins. Comparison of the oligomer symmetry in alphaA-crystallin, HSP 27 and HSP 16.3. J. Biol. Chem. 274, 6 305–6314. Ó FEBS 2002 Similarities between MTB HSP 16.3 and aB-crystallin (Eur. J. Biochem. 269) 1813 . HSP 16. 3. [d,CSalone;, ,HSP1 6 .3: CS (10:1)+1m M KCl and 3. 5 m M MgCl 2 ; s, HSP 16. 3 : CS (10 : 1); . ,HSP 16. 3 : CS (10 : 1) + 3. 5 m M ATPcS; j, HSP 16. 3. Functional similarities between the small heat shock proteins Mycobacterium tuberculosis HSP 16. 3 and human aB-crystallin Melissa