Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.     

Crystallization and preliminary X-ray analysis of an intact soluble-form variant surface glycoprotein from the African trypanosome, Trypanosoma brucei

The intact variant surface glycoprotein (VSG) ILTat 1.24 from Trypanosoma brucei has been crystallized. An amino-terminal domain of the protein comprising two thirds of the sequence had been crystallized previously after proteolytic digestion. Now intact VSG crystals have been grown from 50 mM-Mes (pH 6.5) containing 62% (w/v) saturated ammonium sulfate. The crystals are demonstrated to contain the intact VSG by h.p.l.c. gel filtration and reaction with an antibody to the inositol phosphate oligosaccharide on the VSG carboxy terminus. The space group of the crystals is P6(2)22 (or P6(4)22) with unit cell dimensions a = b = 184 A and c = 214 A. Preparative isoelectric focusing may have facilitated crystallization.     

Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric g protein-coupled signaling to Rho GTPases

BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine nucleotide exchange factors that upregulate Rho GTPases. PDZ-RhoGEF and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to Rho proteins. A approximately 200 residue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with Galpha12/13 proteins. To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human PDZ-RhoGEF. RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine. A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation. A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts. CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions. Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization.     

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.     

Crystallization and preliminary X-ray diffraction studies of dialkylglycine decarboxylase, a decarboxylating transaminase.

The pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase (E.C. 4.1.1.64) has been crystallized by vapor diffusion from a 15% polyethyleneglycol solution with sodium pyruvate as coprecipitant. The space group of the crystals is either P6(2)22 or the enantiomorph, P6(4)22, with one subunit of 46,500 Da per asymmetric unit. The unit cell has dimensions a = b = 152.7 A, c = 86.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and a solvent content of approximately 61%. diffraction extends to 2.3 A resolution.     

Purification, crystallisation and preliminary crystallographic studies of succinate:ubiquinone oxidoreductase from Escherichia coli.

A membrane protein complex, succinate dehydrogenase (SQR) from Escherichia coli has been purified and crystallised. This enzyme is composed of four subunits containing FAD, three iron-sulphur clusters and one haem b as prosthetic groups. The obtained crystals belong to the hexagonal space group P6(3) with the unit-cell dimensions of a=b=123.8 A and c=214.6 A. An asymmetric unit of the crystals contains one SQR monomer (M(r) 120 kDa). A data set is now available at 4.0 A resolution with 88.1% completeness and 0.106 R(merge). We have obtained a molecular replacement solution that shows sensible molecular packing, using the soluble domain of E. coli QFR (fumarate reductase) as a search model. The packing suggests that E. coli SQR is a crystallographic trimer rather than a dimer as observed for the E. coli QFR.     

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).     

Importance of conserved Thr214 in domain A of the Na+,K+ -ATPase for stabilization of the phosphoryl transition state complex in E2P dephosphorylation

Thr(214) of the highly conserved (214)TGES sequence in domain A of the Na(+),K(+)-ATPase was replaced with alanine, and the mutant was compared functionally with the previously characterized domain A mutant Gly(263) --> Ala. Thr(214) --> Ala displayed a conspicuous 150-fold reduction of the apparent vanadate affinity for inhibition of ATPase activity, which could not simply be explained by the observed shifts of the conformational equilibria in favor of E(1) and E(1)P. The intrinsic vanadate affinity of the E(2) form and the effect on the apparent vanadate affinity of displacement of the E(1)-E(2) equilibrium were determined in a phosphorylation assay that allows the enzyme-vanadate complex to be formed under equilibrium conditions. When the E(2) form prevailed, Thr(214) --> Ala retained a reduced vanadate affinity relative to wild type, whereas the affinity of Gly(263) --> Ala became wild type-like. Thus, mutation of Thr(214) affected the intrinsic affinity of E(2) for vanadate. Furthermore, Thr(214) --> Ala showed at least a 5-fold reduced E(2)P dephosphorylation rate relative to wild type in the presence of saturating concentrations of K(+) and Mg(2+). Because vanadate is a phosphoryl transition state analog, it is proposed that defective binding of the phosphoryl transition state complex (transition state destabilization) causes the inability to catalyze E(2)P dephosphorylation properly. By contrast, the phosphorylation site in the E(1) form was unaffected in Thr(214) --> Ala. Replacement of the glutamate, Glu(216), of (214)TGES with alanine was incompatible with cell viability, indicating a very low transport activity or expression level. Our results support the hypothesis that domain A is isolated in the E(1) form, but contributes to make up the catalytic site in the E(2) and E(2)P conformations.     

Crystallization and preliminary X-ray diffraction studies of intact EF-Tu from Thermus aquaticus YT-1

Many attempts have been made to elucidate the three-dimensional structure from elongation factor Tu, but so far the only crystals suitable for X-ray crystallography contained a partially degraded protein. Here, we report the crystallization of a fully active, intact EF-Tu from thermus aquaticus. The crystals belong to hexagonal space group P6(3)(22) and diffract up to 2.6 A. The cell dimensions are a = b = 178 A, c = 238 A and 6 molecules are contained per asymmetric unit.     

Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.     

Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms.

To study the nature of antibody-antigen interactions, we have determined the variable gene sequences of the anti-cytochrome c immunoglobulin G1 (IgG1) monoclonal antibody E8, and obtained diffraction-quality crystals of the E8 antigen-binding fragment (Fab), both free and bound to its antigen, horse cytochrome c. The FabE8 crystals belong to space group P21 with unit cell dimensions of a = 45.0 A, b = 85.1 A, c = 63.3 A and beta = 105.5 degrees, have one FabE8 molecule per asymmetric unit and diffract to at least 2.1 A resolution. Crystals of the FabE8-cytochrome c complex belong to space group P212121 with unit cell dimensions of a = 84.3 A, b = 73.3 A and c = 94.9 A, accommodate one complex per asymmetric unit and diffract to 2.4 A resolution. In the nucleotide-derived amino acid sequences, the light-chain variable domain (VL) but not the heavy-chain variable domain (VH) of E8 is nearly identical to that of the anti-lysozyme antibody D1.3, differing by only five amino acid residues. Only one of these interacts with lysozyme in the D1.3-lysozyme crystal structure. Six negative and four positive charges in the VH complementarity determining regions of E8 complement four positive and three negative charges in the E8 epitope on cytochrome c. These data suggest that only a subset of the residues in an antibody-protein interface may be critical for binding and that the VH may play a dominant role in antigenic recognition.     

Mutation of the highly conserved Arg165 and Glu168 residues of human Gsalpha disrupts the alphaD-alphaE loop and enhances basal GDP/GTP exchange rate

G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Galpha monomer associated with a Gbetagamma heterodimer. Structural studies have shown that Galpha subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the alpha helixD-alpha helixE loop (alphaD-alphaE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Galpha subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the alphaD-alphaE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Galpha subunits, we studied the role of these highly conserved R and E residues in Galpha function. In the present study, we mutated the human Gsalpha R165 and E168 residues to alanine (A), thus generating the R165--> A, E168--> A, and R165/E168--> A mutants. We expressed these human Gsalpha (hGsalpha) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R165/E168--> A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the alphaD-alphaE loop (residues 160-175) and in the GTPaseD at a region required for Gsalpha activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.     

The secreted form of the epidermal growth factor receptor. Characterization and crystallization of the receptor-ligand complex

A protein composed of the external domain of the epidermal growth factor (EGF) receptor is secreted by A431 human tumor cells. The soluble receptor protein was isolated in bulk quantities from cell culture supernatants. It has an intact ligand binding site, exists in a 93-kDa monomeric form, and does not undergo oligomerization upon ligand binding; thus the receptor dimerization reported for the EGF holoreceptor appears not to be a function of its external domain. The unique system of a physiological soluble receptor was utilized for a crystallization study. Crystals were obtained but only in the presence of the ligand. They contained (in equimolar amounts) receptor as well as EGF. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions a = b = 118 A, c = 202 A. The packing density parameter was 3.55 A3/dalton, indicating the asymmetric unit to consist of one receptor-ligand complex.     

Preliminary X-ray crystallographic study of lysozyme produced by Streptomyces globisporus

Lysozyme from Streptomyces globisporus has been crystallized in a form suitable for X-ray structure analysis using ammonium sulfate as a precipitant. The crystals are hexagonal, space group P6(1)22 (P6(5)22) with unit cell dimensions: a = b = 129 A, c = 143 A. There are three or four molecules per asymmetric unit. The crystals diffract X-rays to at least 3.0 A resolution.     

Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.     

Interaction between the catalytic site and the A-M3 linker stabilizes E2/E2P conformational states of Na+,K+-ATPase

The consequences of mutations Ile(265) --> Ala, Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala for the partial reaction steps of the Na(+),K(+)-ATPase transport cycle were analyzed. The mutated residues are part of the long loop ("A-M3 linker") connecting the cytoplasmic A-domain with transmembrane segment M3. It was found that mutation Ile(265) --> Ala displaces the E(1)-E(2) and E(1)P-E(2)P equilibria in favor of E(1)/E(1)P, whereas mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala displace these conformational equilibria in favor of E(2)/E(2)P. The mutations affect both the rearrangement of the cytoplasmic domains (seen by changes in phosphoenzyme properties and apparent ATP/vanadate affinities) and the membrane sector (indicated by change in K(+)/Rb(+) deocclusion rate). Destabilization of E(2)/E(2)P in Ile(265) --> Ala, as well as a direct effect on the intrinsic affinity of the E(2) form for vanadate, may be explained on the basis of the E(2) crystal structures of the Ca(2+)-ATPase, showing interaction of the equivalent isoleucine with conserved residues near the catalytic region of the P-domain. The rate of phosphorylation from ATP was unaffected in Ile(265) --> Ala, indicating a lack of interference with the catalytic function in E(1)/E(1)P. The effects of mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala provide the first evidence in the literature of a relative stabilization of E(2)/E(2)P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E(1)/E(1)P, increased stability of the A3 helix of the A-M3 linker in E(2)/E(2)P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain.     

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.     

Functional consequences of alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal part of transmembrane helix M3 of the Na+,K(+)-ATPase

Mutations Ile279 --> Ala, Ile283 --> Ala, Glu284 --> Ala, His285 --> Ala, His285 --> Lys, His285 --> Glu, Phe286 --> Ala, and His288 --> Ala in transmembrane helix M3 of the Na+,K(+)-ATPase were studied. Except for His285 --> Ala, these mutations were compatible with cell viability, permitting analysis of their effects on the overall and partial reactions of the Na+,K(+)-transport cycle. In Ile279 --> Ala and Ile283 --> Ala, the E1 form accumulated, whereas in His285 --> Lys and His285 --> Glu, E1P accumulated. Phe286 --> Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E2 and E2P, respectively, and showed a unique enhancement of the E1P --> E2P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E1-E2 and E1P-E2P conformational changes. Because the E1-E2 and E1P-E2P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His285 furthermore increased the Na(+)-activated ATPase activity in the absence of K+ ("Na(+)-ATPase activity"). Ile279 --> Ala, Ile283 --> Ala, and His288 --> Ala showed reduced Na+ affinity of the E1 form. The rate of Na(+)-activated phosphorylation from ATP was reduced in Ile279 --> Ala and Ile283 --> Ala, and these mutants showed evidence similar to Glu329 --> Gln of destabilization of the Na(+)-occluded state.