The amino-terminal domain of ClpB supports binding to strongly aggregated proteins

Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpBDeltaN in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpBDeltaN becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpBDeltaN binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.     

Conserved amino acid residues within the amino-terminal domain of ClpB are essential for the chaperone activity

ClpB from Escherichia coli is a member of a protein-disaggregating multi-chaperone system that also includes DnaK, DnaJ, and GrpE. The sequence of ClpB contains two ATP-binding domains that are enclosed between the amino-terminal and carboxyl-terminal regions. The N-terminal sequence region does not contain known functional sequence motifs. Here, we performed site-directed mutagenesis of four polar residues within the N-terminal domain of ClpB (Thr7, Ser84, Asp103 and Glu109). These residues are conserved in several ClpB homologs. We found that the mutations, T7A, S84A, D103A, and E109A did not significantly affect the secondary structure and thermal stability of ClpB, nor did they inhibit the self-association of ClpB, its basal ATPase activity, or the enhanced rate of the ATP hydrolysis by ClpB in the presence of poly-L-lysine. We observed, however, that three mutations, T7A, D103A, and E109A, reduced the casein-induced activation of the ClpB ATPase. The same three mutant ClpB variants also showed low chaperone activity in the luciferase reactivation assay. We found, however, that the four ClpB mutants, as well as the wild-type, bound similar amounts of inactivated luciferase. In summary, we have identified three essential amino acid residues within the N-terminal region of ClpB that participate in the coupling between a protein-binding signal and the ATP hydrolysis, and also support the chaperone activity of ClpB.     

Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization,ATP hydrolysis,and chaperone activity

ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structure-function analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAA-domains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.     

Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains

ClpB is a member of a protein-disaggregating multi-chaperone system in Escherichia coli. The mechanism of protein-folding reactions mediated by ClpB is currently unknown, and the functional role of different sequence regions in ClpB is under discussion. We have expressed and purified the full-length ClpB and three truncated variants with the N-terminal, C-terminal, and a double N- and C-terminal deletion. We studied the protein concentration-dependent and ATP-induced oligomerization of ClpB, casein-induced activation of ClpB ATPase, and ClpB-assisted reactivation of denatured firefly luciferase. We found that both the N- and C-terminal truncation of ClpB strongly inhibited its chaperone activity. The reasons for such inhibition were different, however, for the N- and C-terminal truncation. Deletion of the C-terminal domain inhibited the self-association of ClpB, which led to decreased affinity for ATP and to decreased ATPase and chaperone activity of the C-terminally truncated variants. In contrast, deletion of the N-terminal domain did not inhibit the self-association of ClpB and its basal ATPase activity but decreased the ability of casein to activate ClpB ATPase. These results indicate that the N-terminal region of ClpB may contain a functionally significant protein-binding site, whereas the main role of the C-terminal region is to support oligomerization of ClpB.     

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.     

Mutational analysis of the functional motifs in the ATPase domain of Caenorhabditis elegans fidgetin homologue FIGL-1: firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases

The AAA family proteins usually form a hexameric ring structure. The ATP-binding pocket, which is located at the interface of subunits in the hexamer, consists of three functionally important motifs, the Walker A and B motifs, and the second region of homology (SRH). It is well known that Walker A and B motifs mediate ATP binding and hydrolysis, respectively. Highly conserved arginine residues in the SRH have been proposed to function as arginine fingers, which interact with the gamma-phosphate of bound ATP. To elucidate the mechanism of ATP hydrolysis, we prepared several mutants of the Caenorhabditis elegans fidgetin homologue FIGL-1 carrying a mutation in each of the above-mentioned three motifs. None of the constructed mutants showed ATPase activity. All the mutants except for K362A were able to bind ATP. A decrease in the ATPase activity by mixing wild-type and each mutant subunits was caused by the formation of hetero-hexamers. Mixtures of E416A and R471A, or N461A and R471A led to the formation of hetero-hexamers with partially restored ATPase activities, providing direct, firm evidence for the intersubunit catalysis model. In addition, based on the results obtained with mixtures of K362A with wild-type or R471A subunits, we propose that a conformational change upon ATP binding is required for proper orientation of the arginine fingers, which is essential for efficient hydrolysis of ATP bound to the neighboring subunit.     

Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs: implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of beta-arrestin 2 to self-associate. Amino acids potentially involved in direct protein-protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys(285), Arg(286) and Lys(295) are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into beta-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and beta-arrestin 2-eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both beta-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys(285), Arg(286) and particularly Lys(295) to be important for this interaction. Direct interactions between beta-arrestin 2 and the beta2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of beta-arrestin 2. These were not non-specific effects linked to improper folding of beta-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type beta-arrestin 2, and the interaction of beta-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.     

Two arginines in the cytoplasmic C-terminal domain are essential for voltage-dependent regulation of A-type K+ current in the Kv4 channel subfamily

Contributions of the C-terminal domain of Kv4.3 to the voltage-dependent gating of A-type K+ current (IA) were examined by (i) making mutations in this region, (ii) heterologous expression in HEK293 cells, and (iii) detailed voltage clamp analyses. Progressive deletions of the C terminus of rat Kv4.3M (to amino acid 429 from the N terminus) did not markedly change the inactivation time course of IA but shifted the voltage dependence of steady state inactivation in the negative direction to a maximum of -17 mV. Further deletions (to amino acid 420) shifted this parameter in the positive direction, suggesting a critical role for the domain 429-420 in the voltage-dependent regulation of IA. There are four positively charged amino acids in this domain: Lys423, Lys424, Arg426, and Arg429. The replacement of the two arginines with alanines (R2A) resulted in -23 and -13 mV shifts of inactivation and activation, respectively. Additional replacement of the two lysines with alanines did not result in further shifts. Single replacements of R426A or R429A induced -15 and -10 mV shifts of inactivation, respectively. R2A did not significantly change the inactivation rate but did markedly change the voltage dependence of recovery from inactivation. These two arginines are conserved in Kv4 subfamily, and alanine replacement of Arg429 and Arg432 in Kv4.2 gave essentially the same results. These effects of R2A were not modulated by co-expression of the K+ channel beta subunit, KChIPs. In conclusion, the two arginines in the cytosolic C-terminal domain of alpha-subunits of Kv4 subfamily strongly regulate the voltage dependence of channel activation, inactivation, and recovery.     

Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus

ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and cooperates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-terminal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaperone functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three arginines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP-induced motion of the middle domain independently contribute to the stabilization of the hexamer.     

Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.     

The allosteric regulation of pyruvate kinase

Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit. The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene.     

Multifunctional roles of the conserved Arg residues in the second region of homology of p97/valosin-containing protein

The 97-kDa molecular chaperone valosin-containing protein (VCP) belongs to a highly conserved AAA family and forms a hexameric structure that is essential for its biological functions. The AAA domain contains highly conserved motifs, the Walker A, Walker B, and the second region of homology (SRH). Although Walker A and B motifs mediate ATP binding and hydrolysis, respectively, the function of the SRH in VCP is not clear. We examined the significance of the SRH in VCP, especially the conserved Arg(359) and Arg(362) in the first AAA domain, D1 and Arg(635) and Arg(638) in the second AAA domain, D2. We show that Arg(359) and Arg(362) in D1 are critical for maintaining the hexameric structure and the ability to bind the polyubiquitin chains. Although the rest of the tested SRH mutants retain the hexameric structure, all of them exhibit severely reduced ATPase activity. Tryptophan fluorescence analysis showed that all of the tested mutants can bind to ATP or ADP. Thus, the reduced ATPase activity likely results from the hampered communications among protomers during hydrolysis. Moreover, when the ATPase-defective mutant R635A or R638A is mixed with the Walker A mutant of D2, the ATPase activity is partially restored, suggesting that Arg(635) and Arg(638) can stimulate the ATPase activity of the neighboring protomer. Interestingly, mutation of Arg(359) and Arg(362) uncouples the inhibitory effect of p47, a VCP co-factor, on the ATPase activity of VCP. Therefore, the Arg residues allow D1 to take on a specific conformation that is required for substrate binding and co-factor communications. Taken together, these results demonstrate that the conserved Arg residues in the SRH of both D1 and D2 play critical roles in communicating the conformational changes required for ATP hydrolysis, and SRH in D1 also contributes to substrate binding and co-factor communications.     

Critical role of Lys212 and Tyr140 in porcine NADP-dependent isocitrate dehydrogenase

Lys212 and Tyr140 are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., and Bahnson, B. J. (2002) J. Biol. Chem. 277, 43454-43462). We have constructed mutant enzymes in which Lys212 is replaced by Gln, Tyr, and Arg, and Tyr140 is replaced by Phe, Thr, Glu, and Lys. Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity. At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01-9% of wild type. The most striking change is in the pH-V(max) curves. Wild type depends on the deprotonated form of a group of pKaes 5.7, whereas this pKaes is increased to 7.4 in neutral K212Q and to 8.3 in K212Y. In contrast, the positive K212R has a pKaes of 5.9. These results indicate that (by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex. Lys212 may also stabilize the carbanion formed initially on substrate decarboxylation. The Tyr140 mutants have specific activities at pH 7.4 that are reduced to 0.2-0.5% of those of wild type, whereas their Km values for isocitrate and NADP are not increased. Most notable are the altered pH-V(max) profiles. V(max) is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K. These results suggest that in wild type enzyme, Tyr140 is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes. Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine. These chemical rescue experiments support the conclusion that Tyr140 and Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase.     

Interactions between S4-S5 linker and S6 transmembrane domain modulate gating of HERG K+ channels

Outward movement of the voltage sensor is coupled to activation in voltage-gated ion channels; however, the precise mechanism and structural basis of this gating event are poorly understood. Potential insight into the coupling mechanism was provided by our previous finding that mutation to Lys of a single residue (Asp(540)) located in the S4-S5 linker endowed HERG (human ether-a-go-go-related gene) K(+) channels with the unusual ability to open in response to membrane depolarization and hyperpolarization in a voltage-dependent manner. We hypothesized that the unusual hyperpolarization-induced gating occurred through an interaction between Lys(540) and the C-terminal end of the S6 domain, the region proposed to form the activation gate. Therefore, we mutated six residues located in this region of S6 (Ile(662)-Tyr(667)) to Ala in D540K HERG channels. Mutation of Arg(665), but not the other five residues, prevented hyperpolarization-dependent reopening of D540K HERG channels. Mutation of Arg(665) to Gln or Asp also prevented reopening. In addition, D540R and D540K/R665K HERG reopened in response to hyperpolarization. Together these findings suggest that a single residue (Arg(665)) in the S6 domain interacts with Lys(540) by electrostatic repulsion to couple voltage sensing to hyperpolarization-dependent opening of D540K HERG K(+) channels. Moreover, our findings suggest that the C-terminal ends of S4 and S6 are in close proximity at hyperpolarized membrane potentials.     

Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex

Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.     

Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis

At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp(514), Asp(520), Arg(552), and Arg(611) (numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of (125)I-botrocetin was decreased by mutations at Arg(629), Arg(632), Arg(636), and Lys(667). Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys(599), Arg(629), and Arg(632); among this group the K599A mutant was unique because (125)I-botrocetin binding was normal, suggesting that Lys(599) interacts directly with GPIb. Ristocetin and botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys(534), Arg(571), Lys(572), Glu(596), Glu(613), Arg(616), Glu(626), and Lys(642), whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg(636) and Lys(667). The binding of monoclonal antibody B724 involved Lys(660) and Arg(663), and this antibody inhibits (125)I-botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys(599) on helix 3.     

Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1. Insights into determinants of PDZ domain specificity

We report a structural comparison of the first PDZ domain of ZO-1 (ZO1-PDZ1) and the PDZ domain of Erbin (Erbin-PDZ). Although the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WV(COOH)), that of ZO1-PDZ1 is similar ([R/K/S/T][T/S][W/Y][V/I/L](COOH)) but broadened by increased promiscuity for three of the last four ligand residues. Consequently, the biological function of ZO-1 is also broadened, as it interacts with both tight and adherens junction proteins, whereas Erbin is restricted to adherens junctions. Structural analyses reveal that the differences in specificity can be accounted for by two key differences in primary sequence. A reduction in the size of the hydrophobic residue at the base of the site(0) pocket enables ZO1-PDZ1 to accommodate larger C-terminal residues. A single additional difference alters the specificity of both site(-1) and site(-3). In ZO1-PDZ1, an Asp residue makes favorable interactions with both Tyr(-1) and Lys/Arg(-3). In contrast, Erbin-PDZ contains an Arg at the equivalent position, and this side chain cannot accommodate either Tyr(-1) or Lys/Arg(-3) but, instead, interacts favorably with Glu/Asp(-3). We propose a model for ligand recognition that accounts for interactions extending across the entire binding site but that highlights several key specificity switches within the PDZ domain fold.     

Domain stability in the AAA+ ATPase ClpB from Escherichia coli

ClpB is a heat-shock protein that reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the family of AAA+ ATPases and forms ring-shaped oligomers: heptamers in the absence of nucleotides and hexamers in the presence of nucleotides. We investigated the thermodynamic stability of ClpB in its monomeric and oligomeric forms. ClpB contains six distinct structural domains: the N-terminal domain involved in substrate binding, two AAA+ ATP-binding modules, each consisting of two domains, and a coiled-coil domain inserted between the AAA+ modules. We produced seven variants of ClpB, each containing a single Trp located in each of the ClpB domains and measured the changes in Trp fluorescence during the equilibrium urea-induced unfolding of ClpB. We found that two structural domains: the small domain of the C-terminal AAA+ module and the coiled-coil domain were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation and/or interactions during formation of the ClpB rings.