Distinct domains of Bcl-XL are involved in Bax and Bad antagonism and in apoptosis inhibition

Pro-survival factor Bcl-X(L) can antagonize the pro-apoptotic functions of Bax and Bad via two distinct mechanisms. It can block Bax-mediated cell death by preventing Bax translocation from the cytosol to mitochondria. On the other hand, Bcl-X(L) can neutralize Bad by sequestering it to mitochondria. In order to map the domains of Bcl-X(L) involved in inhibiting Bax and Bad, we have carried out mutational analyses of this protein. This was done by deleting the key domains of Bcl-X(L), including its BH1-4 domains, the flexible loop, the C-terminal hydrophobic domain, and segments of the alpha5-alpha6 hairpin. The resulting Bcl-X(L) mutant constructs were then co-transfected with either GFP-Bax or GFP-Bad. We found that the BH1-4 domains and the C-terminal segment of Bcl-X(L) were essential for blocking Bax localization to mitochondria. On the other hand, only its BH1 and BH3 domains and the C-terminal hydrophobic segment were necessary for sequestering Bad to mitochondria. In addition, by immunoprecipitation analyses, we found that these deletions differentially affected the ability of the Bcl-X(L) mutant proteins to bind Bax and Bad. Finally, cell viability assays indicated that the BH1-4 domains of Bcl-X(L) were the primary domains required for inhibiting staurosporine-induced apoptosis, suggesting that distinct domains of Bcl-X(L) are involved in antagonizing Bax and Bad and in apoptosis inhibition.     

Double point mutant F34W/W140F of staphylococcal nuclease is in a molten globule state but highly competent to fold into a functional conformation

The double point mutant F34W/W140F of staphylococcal nuclease was created and then characterized by far and near-UV CD, size-exclusion chromatography, ANS-binding fluorescence. The results show that this mutant has properties consistent with the classical definition of a molten globule, i.e., substantial secondary structure but no unique tight packing of tertiary structure, a relatively compact size and a larger exposed hydrophobic surface area as compared with the wild type enzyme, indicating that a molten globule can occur under physiological conditions. However, the activity assay showed that the mutant still maintains wild-type levels of activity. To further clarify the mechanism of the substrate-induced reactivation, enzymatic parameters such as K(M)(DNA), K(S)(DNA), K(M)(Ca), K(A)(Ca), K(d)(pdTp), and V(max) were determined, showing that all the parameters of this mutant are similar to those of the wild type enzyme. The results indicate that the F34W/W140F mutant has a similar substrate affinity to the wild type enzyme, and the functional conformation can be restored by substrate binding, which corresponds to the conformational adjustment capability of the mutant upon binding to ligands pdTp and Ca(2+). The severely disrupted tertiary structure and high activity of the mutant indicate that it is highly competent to fold to its functional conformation. The results suggest that the primary structure can only guide the mutant to a molten globule state and that ligand-binding causes the mutant to fold further into its functionally active conformation, indicating that ligand-binding plays an important role in protein folding and catalysis.     

PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells

PUMA is a BH3-only Bcl-2 family protein that plays an essential role in DNA damage-induced apoptosis. PUMA interacts with anti-apoptotic Bcl-2 and Bcl-X(L) and is dependent on Bax to induce apoptosis. In this study, we investigated how the interactions of PUMA with the antiapoptotic proteins coordinate with Bax to initiate apoptosis in HCT116 colon cancer cells. We found that Bcl-X(L) was most effective among several antiapoptotic proteins in suppressing PUMA-induced apoptosis and PUMA-dependent apoptosis induced by the DNA-damaging agent adriamycin. Mutant Bcl-X(L) that cannot interact with Bax was unable to protect cells from PUMA-mediated apoptosis. Knockdown of Bcl-X(L) by RNA interference significantly enhanced PUMA-mediated apoptosis in HCT116 cells but not in PUMA-knockout cells. Furthermore, Bax was found to be dissociated preferentially from Bcl-X(L) in HCT116 cells but not in the PUMA-knockout cells, in response to PUMA induction and adriamycin treatment. PUMA inhibited the association of Bax and Bcl-X(L) in vitro by directly binding to Bcl-X(L) through its BH3 domain. Finally, we found that wild-type Bax, but not mutant Bax deficient in either multimerization or mitochondrial localization, was able to restore PUMA-induced apoptosis in the BAX-knockout cells. Together, these results indicate that PUMA initiates apoptosis in part by dissociating Bax and Bcl-X(L), thereby promoting Bax multimerization and mitochondrial translocation.     

Protein conformational changes in the bacteriorhodopsin photocycle

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation.Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.     

Mitochondrially targeted Bcl-2 and Bcl-X(L) chimeras elicit different apoptotic responses

The Bcl-2 family of proteins interacts at the mitochondria to regulate apoptosis. However, the anti-apoptotic Bcl-2 and Bcl-X(L) are not completely localized to the mitochondria. In an attempt to generate Bcl-2 and Bcl-X(L) chimeras that are constitutively localized to the mitochondria, we substituted their C-terminal transmembrane tail or both the C-terminal transmembrane tail and the adjacent loop with the equivalent regions from Bak or Bax mutant (BaxS184V) as these regions determine the mitochondrial localization of Bak and Bax. The effects of these substitutions on subcellular localization and their activities were assessed following expression in HeLa and CHO K1 cells. The substitution of the C-terminal tail or the C-terminal tail and the adjacent loop of Bcl-2 with the equivalent regions from Bak or the Bax mutant resulted in its association with the mitochondria. This change in subcellular localization of Bcl-2 chimeras triggered cells to undergo apoptotic-like cell death. The localization of this Bcl-2 chimera to the mitochondria may be associated with the disruption of mitochondrial membrane potential. Unlike Bcl-2, the loop structure adjacent to the C-terminal tail in Bcl-X(L) is crucial for its localization. To localize the Bcl-X(L) chimeras to the mitochondria, the loop structure next to the C-terminal tail in Bcl-X(L) protein must remain intact and cannot be substituted by the loop from Bax or Bak. The chimeric Bcl-X(L) with both its C-terminal tail and the loop structure replaced by the equivalent regions of Bak or Bax mutant localized throughout the entire cytosol. The Bcl-X(L) chimeras that are targeted to the mitochondria and the wild type Bcl-X(L) provided same protection against cell death under several death inducing conditions.     

Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2.

Bcl-X(L), a member of the Bcl-2 family, can inhibit many forms of programed cell death. The three-dimensional structure of Bcl-X(L) identified a 60 amino acid loop lacking defined structure. Although amino acid sequence within this region is not conserved among Bcl-2 family members, structural modeling suggested that Bcl-2 also contains a large unstructured region. Compared with the full-length protein, loop deletion mutants of Bcl-X(L) and Bcl-2 displayed an enhanced ability to inhibit apoptosis. Despite enhanced function, the deletion mutants did not have significant alterations in the ability to bind pro-apoptotic proteins such as Bax. The loop deletion mutant of Bcl-2 also displayed a qualitative difference in its ability to inhibit apoptosis. Full-length Bcl-2 was unable to prevent anti-IgM-induced cell death of the immature B cell line WEHI-231. In contrast, the Bcl-2 deletion mutant protected WEHI-231 cells from death. Substantial differences were observed in the ability of WEHI-231 cells to phosphorylate the deletion mutant of Bcl-2 compared with full-length Bcl-2. Bcl-2 phosphorylation was found to be dependent on the presence of an intact loop domain. These results suggest that the loop domain in Bcl-X(L) and Bcl-2 can suppress the anti-apoptotic function of these genes and may be a target for regulatory post-translational modifications.     

Newcastle disease virus HN protein alters the conformation of the F protein at cell surfaces

Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F(0) and F(1) forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.     

Helix Dynamics in LacY: Helices II and IV

Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar and H⁺ binding sites are alternatively accessible to both sides of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within ∼ 4.2 Å of each other. Although a double mutant with Cys replacements at both position 68 and position 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, similar to wild-type LacY; however, strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with results from previous mutagenesis and cross-linking studies, these findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X is also presented.     

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase

Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

Identification of novel isoforms of the BH3 domain protein Bim which directly activate Bax to trigger apoptosis

Bim (Bcl-2-interacting mediator of cell death) is a member of the BH3 domain-only subgroup of Bcl-2 family members, for which three splice variants have been described. Bim is expressed in many healthy cell types, where it is maintained in an inactive conformation through binding to the microtubule-associated dynein motor complex. Upon certain apoptotic stimuli, Bim is released from microtubules and mediates caspase-dependent apoptosis through a mechanism that is still unclear. Here, we have identified and characterized novel splice variants of human Bim mRNA. In particular, we show that a newly discovered, small protein isoform, BimAD, is also able to induce apoptosis strongly in several human cell lines. BimAD and the previously characterized isoform BimS are shown to be capable of heterodimerizing in vivo with both death antagonists (Bcl-2 and Bcl-X(L)) and death agonists (Bax). Mutants of BimAD that bind to Bax but not to Bcl-2 still promote apoptosis, indicating that Bim can regulate apoptosis through direct activation of the Bax-mediated cell death pathway without interaction with antiapoptotic Bcl-2 family members. Furthermore, we have shown that the interaction of the BimS and BimAD isoforms with Bax leads to a conformational change in this protein analogous to that triggered by the BH3-only protein Bid.     

The catalytic and conformational cycle of Aquifex aeolicus KDO8P synthase: role of the L7 loop

KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P(i)). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg(106) side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller K(m)(PEP), larger K(i)(A5P) and K(m)(A5P)) and the product side (smaller K(i)(Pi) and K(i)(KDO8P)) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by P(i) and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.     

Bcl-X(L) and calyculin A prevent translocation of Bax to mitochondria during apoptosis

During many forms of apoptosis, Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the mitochondria and induces cytochrome c release, followed by caspase activation and DNA degradation. Both Bcl-X(L) and the protein phosphatase inhibitor calyculin A have been shown to prevent apoptosis, and here we investigated their impact on Bax translocation. ML-1 cells incubated with either anisomycin or staurosporine exhibited Bax translocation, cytochrome c release, caspase 8 activation, and Bid cleavage; only the latter two events were caspase-dependent, confirming that they are consequences in this apoptotic pathway. Both Bcl-X(L) and calyculin A prevented Bax translocation and cytochrome c release. Bcl-X(L) is generally thought to heterodimerize with Bax to prevent cytochrome c release and yet they remain in different cellular compartments, suggesting that their heterodimerization at the mitochondria is not the primary mechanism of Bcl-X(L)-mediated protection. Using chemical cross-linking agents, Bax appeared to exist as a monomer in undamaged cells. Upon induction of apoptosis, Bax formed homo-oligomers in the mitochondrial fraction with no evidence for cross-linking to Bcl-2 or Bcl-X(L). Considering that both Bcl-X(L) and calyculin A inhibit Bax translocation, we propose that Bcl-X(L) may regulate Bax translocation through modulation of protein phosphatase or kinase signaling.     

c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines

Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unphosphorylated murine type IV c-Abl and measured the kinetic parameters of c-Abl tyrosine kinase activity in a solution with a peptide-based assay. Unphosphorylated c-Abl exhibited substantial peptide kinase activity with K(m) of 204 microm and V(max) of 33 pmol min(-1). Contrary to previous observations using immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K(m) = 91 microm, V(max) = 112 pmol min(-1)). Autophosphorylation stimulated the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates. The autophosphorylation rate was dependent on Abl protein concentration consistent with an intermolecular reaction. A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impaired the activation of wild-type c-Abl by 90% but reduced activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibited the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was minimally activated by autophosphorylation. These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245.     

Binding of enzyme IIAGlc, a component of the phosphoenolpyruvate:sugar phosphotransferase system, to the Escherichia coli lactose permease

Enzyme IIA(Glc), encoded by the crr gene of the phosphoenolpyruvate:sugar phosphotransferase system, plays an important role in regulating intermediary metabolism in Escherichia coli ("catabolite repression"). One function involves inhibition of inducible transport systems ("inducer exclusion"), and with lactose permease, a galactoside is required for unphosphorylated IIA(Glc) binding to cytoplasmic loops IV/V and VI/VII [Sondej, M., Sun, J. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530]. With inside-out membrane vesicles containing the permease, [(125)I]IIA(Glc) binding promoted by melibiose exhibits an affinity (K(D)(IIA)) of approximately 1 microM and a stoichiometry of one mole of IIA(Glc) per six moles of lactose permease. Both the quantity of [(125)I]IIA(Glc) bound and the sugar concentration required for half-maximal IIA(Glc) binding (K(0.5)(IIA)(sug)) was measured for eight permease substrates. Differences in maximal IIA(Glc) binding are observed, and the K(0.5)(IIA)(sug) does not correlate with the affinity of LacY for sugar. Furthermore, K(0.5)(IIA)(sug) does not correlate with sugar affinities for various permease mutants. IIA(Glc) does not bind to a mutant (Cys154 --> Gly), which is locked in an outwardly facing conformation, binds with increased stoichiometry to mutant Lys131 --> Cys, and binds only weakly to two other mutants which appear to be predominantly in either an outwardly or an inwardly facing conformation. When the latter two mutations are combined, sugar-dependent IIA(Glc) binding returns to near wild-type levels. The findings suggest that binding of various substrates to lactose permease results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIA(Glc).     

Identification of the protein-protein contact site and interaction mode of human VDAC1 with Bcl-2 family proteins

Bcl-2 family of proteins plays differential roles in regulation of mitochondria-mediated apoptosis, by either promoting or inhibiting the release of apoptogenic molecules from mitochondria to cytosol. Bcl-2 family proteins modulate the mitochondrial permeability through interaction with adenine nucleotide translocator (ANT), voltage-dependent anion channel (VDAC), ADP/ATP exchange, or oxidative phosphorylation during apoptosis. Although the mitochondrial homeostasis is affected by the relative ratio of pro- and anti-apoptotic Bcl-2 family members, the molecular mechanism underlying the release of mitochondrial intermembrane proteins remains elusive. Here we reported the biochemical evidence that both pro-apoptotic Bax and anti-apoptotic Bcl-X(L) might simultaneously contact the putative loop regions of human VDAC1, and the existence of VDAC1-Bax-Bcl-X(L) tertiary complex in vitro suggested that VDAC1 channel conformation and mitochondrial permeability could be determined by the delicate balance between Bax and Bcl-X(L).     

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.     

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.