Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases.

A number of human diseases have been shown to be associated with mutation in the genes encoding leucine-rich-repeat (LRR)-containing proteins. They include 16 different LRR proteins. Mutations of these proteins are associated with 19 human diseases. The mutations occur frequently within the LRR domains as well as their neighboring domains, including cysteine clusters. Here, based on the sequence analysis of the LRR domains and the known structure of LRR proteins, we describe some features of different sequence variants and discuss their adverse effects. The mutations in the cysteine clusters, which preclude the formation of sulfide bridges or lead to a wrong paring of cysteines in extracellular proteins or extracellular domains, occur with high frequency. In contrast, missense mutations at some specific positions in LRRs are very rare or are not observed at all.

3-Deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli. Model of binding of phosphoenolpyruvate and D-arabinose-5-phosphate.

The crystal structure of 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli was determined by molecular replacement using coordinates given to us by Radaev and co-workers prior to publication. The KDOPS crystals reported by Radaev et al. were grown in the presence of 1.4 M (NH(4))(2)SO(4) and 0.4 M (K/H)(3)PO(4). They are in the cubic space group I23 (a=228.6 A) with a tetramer in the asymmetric unit; the structure has been refined with data to 2.4 A. Our crystals of E. coli KDOPS, grown in 24 % (w/v) polyethylene glycol (PEG) 1500 in the presence of the substrates, 2-phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P), are also in space group I23 (a=118.2 A), with one subunit in the asymmetric unit. The medium of crystallization, 1.8 M SO(4)/PO(4) versus 24 % PEG, does not significantly affect the conformation of KDOPS. The inter-monomer contacts in both structures are the same. The beta(8)/alpha(8) loop (residues 246 to 251) situated near the entrance to the active site is not seen in the 229 A structure but can be traced in the 118 A structure. Most significantly, Radaev et al. interpreted two SO(4)/PO(4) sites in the 229 A structure as marking the phosphate positions of the substrates, PEP and A5P, after the precedent of DAHPS. In the 118 A structure the inner of these two SO(4)/PO(4) peaks is present at the same position as in the 229 A structure of KDOPS. The outer phosphate peak in the 118 A KDOPS is 3.7 A from the outer SO(4)/PO(4) peak in the 229 A structure and is within hydrogen bonding distance of Arg63 of the same subunit and Arg120 of another subunit. Based on the precedent of the d-erythrose-4-phosphate (E4P) modeled in the active site of DAHPS, we have modeled PEP and A5P in KDOPS and compared the coordination of PEP and A5P in KDOPS with that of PEP and E4P in DAHPS.

Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: comparison of the Mn(2+)*2-phosphoglycolate and the Pb(2+)*2-phosphoenolpyruvate complexes and implications for catalysis.

The crystal structure of the phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli in complex with Mn(2+) and the substrate analog, 2-phosphoglycolate (PGL), was determined by molecular replacement using X-ray diffraction data to 2.0 A resolution. DAHPS*Mn*PGL crystallizes in space group C2 (a=210.4 A, b=53.2 A, c=149.4 A, beta=116.1 degrees ) with its four (beta/alpha)(8) barrel subunits related by non-crystallographic 222 symmetry. The refinement was carried out without non-crystallographic symmetry restraints and yielded agreement factors of R=20.9 % and R(free)=23.9 %. Mn(2+), the most efficient metal activator, is coordinated by the same four side-chains (Cys61, His268, Glu302 and Asp326) as is the poorly activating Pb(2+). A fifth ligand is a well-defined water molecule, which is within hydrogen bonding distance to an essential lysine residue (Lys97). The distorted octahedral coordination sphere of the metal is completed by PGL, which replaces the substrate, 2-phosphoenolpyruvate (PEP), in the active site. However, unlike PEP in the Pb*PEP complex, PGL binds the Mn(2+) via one of its carboxylate oxygen atoms. A model of the active site is discussed in which PEP binds in the same orientation as does PGL in the DAHPS*Mn*PGL structure and the phosphate of E4P is tethered at the site of a bound sulfate anion. The re face of E4P can be positioned to interact with the si face of PEP with only small movement of the protein.

Membrane-bound 3D structures reveal the intrinsic flexibility of annexin VI.

Several quasi-ordered arrays and three two-dimensional crystal forms of annexin VI were obtained on artificial lipid monolayers. Three-dimensional reconstructions of the crystal forms exhibit marked differences in the orientations of the two lobes, revealing flexibility of the linker between the two lobes of annexin VI. Evidence is presented that the lobes may bind the monolayer in a parallel orientation, or an antiparallel orientation, in which the second lobe is turned away from the monolayer. It is hypothesized that annexin VI may also adopt several conformations in vivo, underlying different functional roles.

Super-motifs and evolution of tandem leucine-rich repeats within the small proteoglycans--biglycan, decorin, lumican, fibromodulin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin.

Leucine-rich repeats (LRRs) with 20-30 amino acids in unit length are present in many proteins from prokaryotes to eukaryotes. The LRR-containing proteins include a family of nine small proteoglycans, forming three distinct subfamilies: class I contains biglycan/PG-I and decorin/PG-II; class II: lumican, fibromodulin, PRELP, keratocan, and osteoadherin; and class III: epiphycan/PG-Lb and osteoglycin or osteoinductive factor. Comparative sequence analysis of the 34 available protein sequences reveals that these proteoglycans have two types of LRRs, which we call S and T. The type S LRR is 21 residues long and has the consensus sequence of xxaPzxLPxxLxxLxLxxNxI. The type T LRR has 26 residues; its consensus sequence is zzxxaxxxxFxxaxxLxxLxLxxNxL. In both "x" indicates variable residue; "z" is frequently a gap; "a" is Val, Leu, or Ile; and I is Ile or Leu. These type S and TLRRs are ordered into two super-motifs--STT with about 73 residues in classes I and II and ST with about 47 residues in class III. The 12 LRRs in the small proteoglycans of I and II are best represented as (STT)4; the seven LRRs of class III as (ST)T(ST)2. Our analyses indicate that classes I/II and III evolved along different paths after the establishment of the precursor ST, and classes I and II also diverged after the establishment of the precursor (STT)4.

Crystal structure of phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

BACKGROUND: In microorganisms and plants the first step in the common pathway leading to the biosynthesis of aromatic compounds is the stereospecific condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P) giving rise to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). This reaction is catalyzed by DAHP synthase (DAHPS), a metal-activated enzyme, which in microorganisms is the target for negative-feedback regulation by pathway intermediates or by end products. In Escherichia coli there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids. RESULTS: The crystal structure of the phenylalanine-regulated form of DAHPS complexed with PEP and Pb2+ (DAHPS(Phe)-PEP-Pb) was determined by multiple wavelength anomalous dispersion phasing utilizing the anomalous scattering of Pb2+. The tetramer consists of two tight dimers. The monomers of the tight dimer are coupled by extensive interactions including a pair of three-stranded, intersubunit beta sheets. The monomer (350 residues) is a (beta/alpha)8 barrel with several additional beta strands and alpha helices. The PEP and Pb2+ are at the C-ends of the beta strands of the barrel, as is SO4(2-), inferred to occupy the position of the phosphate of E4P. Mutations that reduce feedback inhibition cluster about a cavity near the twofold axis of the tight dimer and are centered approximately 15 A from the active site, indicating the location of a separate regulatory site. CONCLUSIONS: The crystal structure of DAHPS(Phe)-PEP-Pb reveals the active site of this key enzyme of aromatic biosynthesis and indicates the probable site of inhibitor binding. This is the first reported structure of a DAHPS; the structure of its two paralogs and of a variety of orthologs should now be readily determined by molecular replacement.

Contributions to maxima in protein kinase C activation.

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.

Crystal structure of bovine annexin VI in a calcium-bound state.

The crystal structure of a calcium-bound form of bovine annexin VI has been determined with X-ray diffraction data to 2.9 A by molecular replacement. Six Ca2+ ions were found, five in AB loops, one in a DE loop. Two loops (II-AB, which binds calcium, and V-AB, which does not) have conformations that differ significantly from those in calcium-free, human recombinant annexin VI. There are only small differences between the calci- and the apo-annexin VI in the rest of the molecule. Calcium by itself does not promote a major conformational change.

Two-dimensional crystals of protein kinase C.

Three two-dimensional (2D) crystal forms of protein kinase C (PKC) alpha and three of PKC delta have been grown on lipid monolayers composed of dioleoylphosphatidylcholine: dioleoylphosphatidylserine: (45:50:5 molar ratio). In the absence of DO, two additional 2D crystals of PKC delta are seen, suggesting that the presence of diolein (DO) alters the conformation of intact PKC at the lipid surface. Reconstructions of electron micrographs of these eight lattices show good reproducibility and indicate that several are appropriate for three-dimensional reconstruction to 20 A resolution.

Classification and evolution of EF-hand proteins.

Forty-five distinct subfamilies of EF-hand proteins have been identified. They contain from two to eight EF-hands that are recognizable by amino acid sequence as being statistically similar to other EF-hand domains. All proteins within one subfamily are congruent to one another, i.e. the dendrogram computed from one of the EF-hand domains is similar, within statistical error, to the dendrogram computed from another(s) domain. Thirteen subfamilies--including Calmodulin, Troponin C, Essential light chain, Regulatory light chain--referred to collectively as CTER, are congruent with one another. They appear to have evolved from a single ur-domain by two cycles of gene duplication and fusion. The subfamilies of CTER subsequently evolved by gene duplications and speciations. The remaining 32 subfamilies do not show such general patterns of congruence; however, some--such as S100, intestinal calcium binding protein (calbindin 9 kd), and trichohylin--do not form congruent clusters of subfamilies. Nearly all of the domains 1, 3, 5, and 7 are most similar to other ODD domains. Correspondingly the EVEN numbered domains of all 45 subfamilies most closely resemble EVEN domains of other subfamilies. Many sequence and chemical characteristics do not show systemic trends by subfamily or species of host organisms; such homoplasy is widespread. Eighteen of the subfamilies are heterochimeric; in addition to multiple EF-hands they contain domains of other evolutionary origins.

The crystal structure of annexin VI indicates relative rotation of the two lobes upon membrane binding.

The crystal structure of bovine liver annexin VI has been determined to low resolution by molecular replacement. The first lobe (domains 1-4) is rotated about 90 degrees relative to the second lobe (domains 5-8). Since the same crystal form (P4(3), 68 X 68 X 205 A) grew from (NH4)2SO4, polyethylene glycol, and sodium acetate with and without added calcium, this probably reflects the structure in solution. When bound to a lipid monolayer both lobes of annexin VI are coplanar. This implies a significant change in conformation upon binding to membranes.

The linker of calmodulin lacking Glu84 is elongated in solution, although it is bent in the crystal.

The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 A and 62.5 A, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 A and 55.0 A, and smaller than those expected from the crystal structure of wild type, 22.8 A and 67.5 A. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether.

Purification, crystallization, and preliminary crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The phenylalanine-regulated isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli, its binary complexes with either substrate, phosphoenolpyruvate (PEP), or feedback inhibitor, Phe, and its ternary complexes with either PEP or Phe plus metal cofactor (either Mn2+, Cd2+, or Pb2+) were crystallized from polyethylglycol (PEG) solutions. All crystals of the DAHPS without Phe belong to space group C2, with cell parameters a = 213.5 A, b = 54.3 A, c = 149.0 A, beta = 116.6 degrees. All crystals of the enzyme with Phe also belong to space group C2, but with cell parameters a = 297.1 A, b = 91.4 A, c = 256.5 A, and beta = 148.2 degrees.

Crystallization and preliminary crystallographic studies of 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli.

3-Deoxy-D-manno-octulosonate-8-phosphate (KDOP) synthase catalyzes the production of KDOP from phosphoenolpyruvate (PEP) and arabinose-5-phosphate (A5P). In gram-negative bacteria KDOP is subsequently dephosphorylated, cytidylylated, and linked to lipid A and is required for lipid A incorporation into the outer membrane (Raetz, Annu. Rev. Biochem. 59:129-170, 1990). We have crystallized two forms of KDOP synthase belonging to space groups I23 or I2(1)3, one with a = b = c = 118.0 A and the other with a = b = c = 233 A.

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa.

The proteins, AlgR3 and AlgP, are involved in the regulation of alginate synthesis in Pseudomonas. They contain multiple repeats of Ala*Ala*Lys*Pro as do several other proteins that resemble histones. The interactions of synthesis oligopeptides composed of repeated Ala*Ala*Lys*Pro or Lys*Lys*Ser*Pro units with DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl) group attached to the N-termini of the peptides. DNA quenching of the Fmoc fluorescence of the peptides was used to estimate the apparent association constants for the interaction of Fmoc(AAKP)nOH (n = 2, 4, 8, 18, 32) and of Fmoc (KKSP)nOH (n = 2, 4, 8, 16, 20, 32) with DNA. The Fmoc(AAKP)nOH peptides bind to DNA only at low ionic strength; the Fmoc(KKSP)n OH peptides interact with DNA at both low (0.05 M KCl) and high (0.2 M KCl) salt. At low ionic strength an increase in the number of the repeat units causes an increase in the apparent association constant up to approximately 2 x 10(6) M-1 for both types of peptides at N congruent to 24. The insertion of an AAKTA unit into the middle of the Fmoc(AAKP)8OH peptide increases its affinity to DNA. We propose a model of (AAKP)n and of its interaction with DNA. The repeat unit consists of a single turn of alpha-helix followed by a bend necessitated by Pro. The resultant coiled-coil forms a right-handed superhelix with 10 AAKPs per repeat distance of approximately 33 A.(ABSTRACT TRUNCATED AT 250 WORDS)

p-Chlorotetrafluorophenyl esters of N-protected amino acids.

p-Chlorotetrafluorophenyl (Tfc) esters of protected amino acids and peptides are more reactive than are the well known pentafluorophenyl (Pfp) esters. Two reagents, p-chlorotetrafluorophenyltrifluoroacetate (Tfc-OTfa) and di-(p-chlorotetrafluorophenyl)carbonate (di-Tfc-carbonate), can be used for their syntheses, thereby avoiding use of the allergic dicyclohexylcarbodiimide. This is especially important for bulk preparations. Many Fmoc- and Boc-amino acid-OTfc esters have been synthesized and characterized. The hexadecameric tandem repeat H-(AlaAlaLysPro)4-OH was synthesized using di-Tfc-carbonate for the preparation of Tfc-esters.

Crystal structure of human class mu glutathione transferase GSTM2-2. Effects of lattice packing on conformational heterogeneity.

The structures of three crystal forms of the class mu human glutathione transferase GSTM2-2 have been determined. X-ray phase information was obtained independently from molecular replacement and from anomalous scattering by a single isomorphous derivative. One crystal form contains a single monomer in the asymmetric unit and has been refined to 1.85 A with an overall R factor of 22.6%. The second form contains a single dimer in the asymmetric unit and has been refined to 3.5 A with an R factor of 20.7%. The third form contains two dimers in the asymmetric unit and has been refined to 3.0 A with an R factor of 25.0%. Although all three crystal forms were grown from solutions that contained glutathione-dinitrobenzene, electron density can only be seen for the glutathione portion of the ligand. The first 202 residues in the seven crystallographically independent monomers of GSTM2-2 are essentially identical in structure. However, heterogeneity in the conformation of the side-chain of Tyr115 is observed in the different monomers. The tertiary structure of residues 1-202 is similar to that of the corresponding region in the class mu isoform of glutathione transferase from rat, GST3-3 (Ji et al. (1992), Biochemistry, 31, 10169-10184). However, significant differences in the conformation of the two enzymes have been observed in the region of the active site that binds hydrophobic substrates. These differences include a 2 A shift in the carboxy terminus of a helix, and significant heterogeneity in the conformation of the last 15 residues of the carboxy terminus. The conformation and degree of disorder of the last 15 residues correlates with the extent of protein-protein contacts within the unit cell.