Phosphorylated and non-phosphorylated HCK kinase domains produced by cell-free protein expression.

Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant proteins easily. However, especially in the case of protein kinase, product solubility remains one of the major challenges. To overcome this problem, we prepared a chaperone-supplemented extract from Escherichia coli BL21 cells harboring a plasmid encoding a set of chaperone genes, dnaK, dnaJ, and grpE. We explored cell-disruption procedures and constructed an efficient protein synthesis system. Employing this system, we produced the kinase domain of human hematopoietic cell kinase (HCK) to obtain further structural information about its molecular interaction with one of its inhibitors, previously developed by our group (RK-20449). Lower reaction temperature improved the solubility, and addition of a protein phosphatase (YpoH) facilitated the homogeneous production of the non-phosphorylated kinase domain. Crystals of the purified product were obtained and the kinase-inhibitor complex structure was solved at 1.7 Šresolution. In addition, results of kinase activity measurement, using a synthetic substrate, showed that the kinase activity was facilitated by autophosphorylation at Tyr416, as confirmed by the peptide mass mapping.

Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel ß4 subunit explain its role in cell-cell adhesion.

Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary ß subunits, designated as ß1/ß1B-ß4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the ß4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the ß4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys58) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35). Under reducing conditions, an N-terminally deleted ß4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the ß4 cis dimer contributes to the trans homophilic interaction of ß4 in cell-cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of ß4 affects α-ß4 complex formation. These observations provide the structural basis for the parallel dimer formation of ß4 in VGSCs and reveal its mechanism in cell-cell adhesion.

Targeting Ras-Driven Cancer Cell Survival and Invasion through Selective Inhibition of DOCK1.

Oncogenic Ras plays a key role in cancer initiation but also contributes to malignant phenotypes by stimulating nutrient uptake and promoting invasive migration. Because these latter cellular responses require Rac-mediated remodeling of the actin cytoskeleton, we hypothesized that molecules involved in Rac activation may be valuable targets for cancer therapy. We report that genetic inactivation of the Rac-specific guanine nucleotide exchange factor DOCK1 ablates both macropinocytosis-dependent nutrient uptake and cellular invasion in Ras-transformed cells. By screening chemical libraries, we have identified 1-(2-(3'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-oxoethyl)-5-pyrrolidinylsulfonyl-2(1H)-pyridone (TBOPP) as a selective inhibitor of DOCK1. TBOPP dampened DOCK1-mediated invasion, macropinocytosis, and survival under the condition of glutamine deprivation without impairing the biological functions of the closely related DOCK2 and DOCK5 proteins. Furthermore, TBOPP treatment suppressed cancer metastasis and growth in vivo in mice. Our results demonstrate that selective pharmacological inhibition of DOCK1 could be a therapeutic approach to target cancer cell survival and invasion.

SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity.

The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4-P1 and P1'. In this study, we determined the crystal structure of 3CLpro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3' position [Phe(P3')] is snugly accommodated in the S3' pocket. Mutations of Phe(P3') impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3' subsite is formed by Phe(P2)-induced conformational changes of 3CLpro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3' subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3'). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3') is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).

A redox switch shapes the Lon protease exit pore to facultatively regulate proteolysis.

The Lon AAA+ protease degrades damaged or misfolded proteins in its intramolecular chamber. Its activity must be precisely controlled, but the mechanism by which Lon is regulated in response to different environments is not known. Facultative anaerobes in the Enterobacteriaceae family, mostly symbionts and pathogens, encounter both anaerobic and aerobic environments inside and outside the host's body, respectively. The bacteria characteristically have two cysteine residues on the Lon protease (P) domain surface that unusually form a disulfide bond. Here we show that the cysteine residues act as a redox switch of Lon. Upon disulfide bond reduction, the exit pore of the P-domain ring narrows by ∼30%, thus interrupting product passage and decreasing activity by 80%; disulfide bonding by oxidation restores the pore size and activity. The redox switch (E°' = -227 mV) is appropriately tuned to respond to variation between anaerobic and aerobic conditions, thus optimizing the cellular proteolysis level for each environment.

HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-ß type I receptor.

Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-ß and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-ß2 in (CAGA)9-Luc CCL64 cells and in human hepatic cell lines via binding to TGF-ß type I receptor (TßRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TßRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TßRI blocked the TGF-ß mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-ß2 and functions, at least in part, via directly binding to and activating TßRI, thereby enhancing liver fibrosis.

Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 µM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 µM ATP (<>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

Autoprocessing mechanism of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CLpro) from its polyproteins.

Like many other RNA viruses, severe acute respiratory syndrome coronavirus (SARS-CoV) produces polyproteins containing several non-structural proteins, which are then processed by the viral proteases. These proteases often exist within the polyproteins, and are excised by their own proteolytic activity ('autoprocessing'). It is important to investigate the autoprocessing mechanism of these proteases from the point of view of anti-SARS-CoV drug design. In this paper, we describe a new method for investigating the autoprocessing mechanism of the main protease (M(pro)), which is also called the 3C-like protease (3CL(pro)). Using our method, we measured the activities, under the same conditions, of the mature form and pro-forms with the N-terminal pro-sequence, the C-terminal pro-sequence or both pro-sequences, toward the pro-form with both N- and C-terminal pro-sequences. The data indicate that the pro-forms of the enzyme have proteolytic activity, and are stimulated by the same proteolytic activity. The stimulation occurs in two steps, with approximately eightfold stimulation by N-terminal cleavage, approximately fourfold stimulation by C-terminal cleavage, and 23-fold stimulation by the cleavage of both termini, compared to the pro-form with both the N- and C-terminal pro-sequences. Such cleavage mainly occurs in a trans manner; i.e. the pro-form dimer cleaves the monomeric form. The stimulation by N-terminal pro-sequence removal is due to the cis (intra-dimer and inter-protomer) effect of formation of the new N-terminus, whereas that by C-terminal cleavage is due to removal of its trans (inter-dimer) inhibitory effect. A numerical simulation of the maturation pathway is presented.

Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA.

In thermophilic bacteria, specific 2-thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2-thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur-carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2-thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N- and C-terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA(Ile)2 lysidine synthetase), than to the other type of tRNA 2-thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP-binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA-binding site.

Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.

In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. The hydrophilic V(1) portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A(3)B(3) complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V(1)-ATPase from the A(3)B(3) and DF complexes. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A(3)B(3) complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 Å) and nucleotide-bound (2.7 Å) V(1)-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V(1)-ATPase.

Structure of the Rho-specific guanine nucleotide-exchange factor Xpln.

Xpln is a guanine nucleotide-exchange factor (GEF) for Rho GTPases. A Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain is a widely adopted GEF-domain architecture. The Xpln structure solely comprises these two domains. Xpln activates RhoA and RhoB, but not RhoC, although their GTPase sequences are highly conserved. The molecular mechanism of the selectivity of Xpln for Rho GTPases is still unclear. In this study, the crystal structure of the tandemly arranged DH-PH domains of mouse Xpln, with a single molecule in the asymmetric unit, was determined at 1.79 Šresolution by the multiwavelength anomalous dispersion method. The DH-PH domains of Xpln share high structural similarity with those from neuroepithelial cell-transforming gene 1 protein, PDZ-RhoGEF, leukaemia-associated RhoGEF and intersectins 1 and 2. The crystal structure indicated that the α4-α5 loop in the DH domain is flexible and that the DH and PH domains interact with each other intramolecularly, thus suggesting that PH-domain rearrangement occurs upon RhoA binding.

Structural insight into the interaction of ADP-ribose with the PARP WWE domains.

The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains.

Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms.

DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokinesis 2-engulfment and cell motility protein 1 (DOCK2•ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Šresolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2•ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2•ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2•ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.

Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3.

Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430-457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90-120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3(2)12, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å(3) Da(-1) and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution.

Crystal structures of the armadillo repeat domain of adenomatous polyposis coli and its complex with the tyrosine-rich domain of Sam68.

Adenomatous polyposis coli (APC) is a tumor suppressor protein commonly mutated in colorectal tumors. APC plays important roles in Wnt signaling and other cellular processes. Here, we present the crystal structure of the armadillo repeat (Arm) domain of APC, which facilitates the binding of APC to various proteins. APC-Arm forms a superhelix with a positively charged groove. We also determined the structure of the complex of APC-Arm with the tyrosine-rich (YY) domain of the Src-associated in mitosis, 68 kDa protein (Sam68), which regulates TCF-1 alternative splicing. Sam68-YY forms numerous interactions with the residues on the groove and is thereby fixed in a bent conformation. We assessed the effects of mutations and phosphorylation on complex formation between APC-Arm and Sam68-YY. Structural comparisons revealed different modes of ligand recognition between the Arm domains of APC and other Arm-containing proteins.

Inhibitor-bound structures of human pyruvate dehydrogenase kinase 4.

The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDC activity is tightly regulated by four members of a family of pyruvate dehydrogenase kinase isoforms (PDK1-4), which phosphorylate and inactivate PDC. Recently, the development of specific inhibitors of PDK4 has become an especially important focus for the pharmaceutical management of diabetes and obesity. In this study, crystal structures of human PDK4 complexed with either AMPPNP, ADP or the inhibitor M77976 were determined. ADP-bound PDK4 has a slightly wider active-site cleft and a more disordered ATP lid compared with AMPPNP-bound PDK4, although both forms of PDK4 assume open conformations with a wider active-site cleft than that in the closed conformation of the previously reported ADP-bound PDK2 structure. M77976 binds to the ATP-binding pocket of PDK4 and causes local conformational changes with complete disordering of the ATP lid. M77976 binding also leads to a large domain rearrangement that further expands the active-site cleft of PDK4 compared with the ADP- and AMPPNP-bound forms. Biochemical analyses revealed that M77976 inhibits PDK4 with increased potency compared with the previously characterized PDK inhibitor radicicol. Thus, the present structures demonstrate for the first time the flexible and dynamic aspects of PDK4 in the open conformation and provide a basis for the development of novel inhibitors targeting the nucleotide-binding pocket of PDK4.

Crystal structure of Sulfolobus tokodaii Sua5 complexed with L-threonine and AMPPNP.

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N(6)-threonylcarbamoyl moiety is composed of L-threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L-threonine-binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L-threonine more strongly than L-serine and glycine. The Kd values of Sua5 for L-threonine and L-serine are 9.3 µM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L-threonine, at 1.8 Å resolution. The L-threonine is bound next to AMPPNP in the same pocket of the N-terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine-specific recognition. The carboxyl group and the side-chain hydroxyl and methyl groups of L-threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L-threonine is located in a suitable position to react together with ATP for the synthesis of N(6)-threonylcarbamoyladenosine.

Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na+-ATPase.

Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V(1); NtpA(3)-B(3)-D-G) and an integral membrane domain (V(0); NtpI-K(10)) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V(1) portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 microM for ADP or 3.1 microM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA(3)-B(3) heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA(3)-B(3) forming V(1) (NtpA(3)-B(3)-D-G) complex independent of nucleotides. The V(1) formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V(1) complex was as high as that of native V(1)-ATPase purified from the V(0)V(1) complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V(1) complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V(1)-ATPase complex.

Crystal structure of epstein-barr virus DNA polymerase processivity factor BMRF1.

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-DeltaC) was solved in an oligomeric state. The molecular structure of BMRF1-DeltaC shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-DeltaC, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-DeltaC exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-DeltaC associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-DeltaC probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-DeltaC is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.

Novel dimerization mode of the human Bcl-2 family protein Bak, a mitochondrial apoptosis regulator.

Interactions of Bcl-2 family proteins play a regulatory role in mitochondrial apoptosis. The pro-apoptotic protein Bak resides in the outer mitochondrial membrane, and the formation of Bak homo- or heterodimers is involved in the regulation of apoptosis. The previously reported structure of the human Bak protein (residues Glu16-Gly186) revealed that a zinc ion was coordinated with two pairs of Asp160 and His164 residues from the symmetry-related molecules. This zinc-dependent homodimer was regarded as an anti-apoptotic dimer. In the present study, we determined the crystal structure of the human Bak residues Ser23-Asn185 at 2.5A, and found a distinct type of homodimerization through Cys166 disulfide bridging between the symmetry-related molecules. In the two modes of homodimerization, the molecular interfaces are completely different. In the membrane-targeted model of the S-S bridged dimer, the BH3 motifs are too close to the membrane to interact directly with the anti-apoptotic relatives, such as Bcl-x(L). Therefore, the Bak dimer structure reported here may represent a pro-apoptotic mode under oxidized conditions.