The glucose transporter of the Escherichia coli phosphotransferase system: linker insertion mutants and split variants

The IICB(Glc) subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate. It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus. Random DNase I cleavage, EcoRI linker insertion, and screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity. They carried inserts of up to 29 residues and short deletions in periplasmic loops 1, 2, and 3, in the long cytoplasmic loop 3, and in the linker region between the membrane spanning IIC(Glc) and the cytoplasmic IIB(Glc) domains. Disruption of the gene at the sites of linker insertion decreased the expression level and diminished phosphotransferase activity to between 7% and 32%. IICB(Glc) with a discontinuity in the cytoplasmic loop was purified to homogeneity as a stable complex. It was active only if encoded by a dicistronic operon but not if encoded by two genes on two different replicons, suggesting that spatial proximity of the nascent polypeptide chains is important for folding and membrane assembly.     

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB(Glc) subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB(Glc) are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB(Glc) and IIA(Glc) were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA(Glc) and IICB(Glc) had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding.     

Transient state kinetics of enzyme IICBGlc, a glucose transporter of the phosphoenolpyruvate phosphotransferase system of Escherichia coli: equilibrium and second order rate constants for the glucose binding and phosphotransfer reactions

During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA(Glc) and Enzyme IICB(Glc), the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA(Glc) to IICB(Glc) to Glc) and also the second order rate constants for the transfer from phospho-IIA(Glc) to molecularly cloned IIB(Glc), the soluble, cytoplasmic domain of IICB(Glc). The rate constants for the forward and reverse phosphotransfer reactions between IIA(Glc) and IICB(Glc) were 3.9 x 10(6) and 0.31 x 10(6) m(-1) s(-1), respectively, and the rate constant for the physiologically irreversible reaction between [P]IICB(Glc) and Glc was 3.2 x 10(6) m(-1) s(-1). From the rate constants, the equilibrium constants for the transfer of the phospho-group from His90 of [P]IIA(Glc) to the phosphorylation site Cys of IIB(Glc) or IICB(Glc) were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB(Glc) were invariably found to contain endogenous, firmly bound Glc (estimated K'(D) approximately 10(-7) m). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB(Glc) has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.     

Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICB(Glc) (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc). We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside. Binding of Mlc to Enzyme IICB(Glc) in vitro required the IIB domain and the IIC-B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross-dephosphorylation of IIB(Glc) during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.     

Preliminary crystallographic data and quaternary structural implications of the central subunit of the multi-subunit complex transcarboxylase

The hexameric central subunit (Mr = 360,000) of the multi-subunit complex transcarboxylase has been crystallized by bulk dialysis against 250 mM-sodium acetate (pH 5.5). The crystals are cubic, a = 193.1 A, space group P4(1)32 or enantiomorph. The number of molecules per unit cell is four and was deduced from the density of the crystals (1.10 g cm-3) and the mother liquor (1.01 g cm-3) and the specific volume of the protein calculated from molecular dimensions obtained from electron microscopy studies. Four molecules per cell requires the central subunits to lie on 3-fold axes, which are perpendicular to 2-fold rotation axes, so that the molecules satisfy 32 symmetry giving one subunit as the asymmetric unit. Of the four possible models that have been considered for the quaternary structure of transcarboxylase, only that with antiparallel subunits, two sets of isologous binding sites and D3 symmetry is in agreement with the symmetry requirements of the cubic crystals.     

Genetic requirements for growth of Escherichia coli K12 on methyl-alpha-D-glucopyranoside and the five alpha-D-glucosyl-D-fructose isomers of sucrose

Strains of Escherichia coli K12, including MG-1655, accumulate methyl-alpha-D-glucopyranoside via the phosphoenolpyruvate-dependent glucose:phosphotransferase system (IICB(Glc)/IIA(Glc)). High concentrations of intracellular methyl-alpha-D-glucopyranoside 6-phosphate are toxic, and cell growth is prevented. However, transformation of E. coli MG-1655 with a plasmid (pAP1) encoding the gene aglB from Klebsiella pneumoniae resulted in excellent growth of the transformant MG-1655 (pAP1) on the glucose analog. AglB is an unusual NAD+/Mn2+-dependent phospho-alpha-glucosidase that promotes growth of MG-1655 (pAP1) by catalyzing the in vivo hydrolysis of methyl-alpha-D-glucopyranoside 6-phosphate to yield glucose 6-phosphate and methanol. When transformed with plasmid pAP2 encoding the K. pneumoniae genes aglB and aglA (an alpha-glucoside-specific transporter AglA (IICB(Agl))), strain MG-1655 (pAP2) metabolized a variety of other alpha-linked glucosides, including maltitol, isomaltose, and the following five isomers of sucrose: trehalulose alpha(1-->1), turanose alpha(1-->3), maltulose alpha(1-->4), leucrose alpha(1-->5), and palatinose alpha(1-->6). Remarkably, MG-1655 (pAP2) failed to metabolize sucrose alpha(1-->2). The E. coli K12 strain ZSC112L (ptsG::cat manXYZ nagE glk lac) can neither grow on glucose nor transport methyl-alpha-D-glucopyranoside. However, when transformed with pTSGH11 (encoding ptsG) or pAP2, this organism provided membranes that contained either the PtsG or AglA transporters, respectively. In vitro complementation of transporter-specific membranes with purified general phosphotransferase components showed that although PtsG and AglA recognized glucose and methyl-alpha-D-glucopyranoside, only AglA accepted other alpha-D-glucosides as substrates. Complementation experiments also revealed that IIA(Glc) was required for functional activity of both PtsG and AglA transporters. We conclude that AglA, AglB, and IIA(Glc) are necessary and sufficient for growth of E. coli K12 on methyl-alpha-D-glucoside and related alpha-D-glucopyranosides.     

X-ray diffraction of heavy-atom labelled two-dimensional crystals of rhodopsin identifies the position of cysteine 140 in helix 3 and cysteine 316 in helix 8

We have used site-specific heavy-atom labelling and X-ray diffraction to localize single amino acid residues in the cytoplasmic domain of the integral membrane protein rhodopsin, the dim-light photoreceptor of retinal vertebrate rod cells. Two-dimensional orthorhombic crystals of the space group p22(1)2(1) (a=59.5(+/-1) A and b=82.7(+/-1.5) A) were produced from detergent-solubilized, partially delipidated rhodopsin. To obtain milligram amounts of two-dimensional crystals, which are required for X-ray diffraction, the yield of the crystalline material was significantly increased by reconstitution of rhodopsin in the presence of cholesterol (1:2 to 1:10 mol/mol) and by adding polar organic solvents to the dialysis buffer. The native cysteine residues C140 and C316 were then selectively labelled with mercury using the sulphydryl-specific reagent p-chloromercuribenzoate (1.6-2.1 mol Hg per mol rhodopsin). The labelling did not affect the unit cell dimensions. Optical absorption spectra of labelled and native two-dimensional rhodopsin crystals showed the characteristic 11-cis-retinal peak at 498 nm, which corresponds to the dark state of rhodopsin. The in-plane position of the mercury label was calculated at 9.5 A resolution from the intensity differences in the X-ray diffraction patterns of labelled and native crystals using Fourier difference methods and the phase information from electron crystallography. The label positions were in excellent agreement with the positions of C140 at the cytoplasmic end of helix 3 and of C316 in the cytoplasmic helix 8 recently obtained from three-dimensional rhodopsin crystals. Whereas these high-resolution diffraction studies were performed under cryogenic conditions (100 K), our results were obtained at room temperature with fully hydrated membranes and in the absence of loop-loop crystal contacts. To study the structural changes of the cytoplasmic loops involved in activation and signal transduction, our more physiological conditions offer important advantages. Furthermore, the localization of C316 is the first direct proof that the electron density on top of helix 1 observed by cryo-electron microscopy is a part of the C-terminal loop. Our approach is of particular interest for investigations of other membrane proteins, for which 3D crystals are not available. Structural constraints from heavy-atom labels at strategic sites enable the assignment of a position in the amino acid sequence to features visible in a low-resolution density map and the study of conformational changes associated with different functional states of the membrane protein.     

CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals

The structure of Na, K-ATPase was determined by electron crystallography at 9.5 A from multiple small 2-D crystals induced in purified membranes isolated from the outer medulla of pig kidney. The density map shows a protomer stabilized in the E(2) conformation which extends approximately 65 A x 75 A x 150 A in the asymmetric unit of the P2 type unit cell. The alpha, beta, and gamma subunits were demonstrated in the membrane crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains, which are similar in overall shape to the domains of the calcium pump of the sarcoplasmic reticulum. One of these domains gives rise to a characteristic elongated projection onto the membrane plane while the putative nucleotide binding and phosphorylation domains form comparatively compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit and is located as an extension of the transmembrane region perpendicular to the membrane plane. The structure of the lipid bilayer spanning part suggests the positions for the transmembrane helix from the beta subunit as well as the small gamma subunit present in this Na,K-ATPase. Two groups of ten helices from the catalytic alpha subunit corresponds to the remaining density in the transmembrane region. The present results demonstrate distinct similarities between the structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy and the reported X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmatic domains.     

Crystallization and crystallographic characterization of the iron-sulfur-containing DNA-repair enzyme endonuclease III from Escherichia coli.

Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities. It could serve to repair damaged thymine residues in DNA via base excision-repair. We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals. Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A. A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence. The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme. The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins.     

Intein-mediated cyclization of a soluble and a membrane protein in vivo: function and stability

Cyclized subunits of the E. coli glucose transporter were produced in vivo by intein mediated trans-splicing. IIA(Glc) is a beta-sandwich protein, IICB(Glc) spans the membrane eight times. Genes encoding the circularly permuted precursors U(Cdelta)-IIA(Glc)-U(Ndelta) and U(Cdelta)-IICB(Glc)-U(Ndelta) were assembled from DNA fragments encoding the 3' and 5' segments of the recA intein of M. tuberculosis and crr and ptsG of E. coli, respectively. A 20-residues long, Ala-Pro rich linker peptide and/or a histidine tag were used to join the native N- and C-termini in the cyclized proteins. The cyclized proteins complemented growth of glucose auxotrophic strains. Purified, cyclized IIA(Glc) and IICB(Glc) had 100 and 25%, respectively, of wild-type glucose phosphotransferase activity. They had an increased electrophoretic mobility, which decreased upon linearization of the proteins with chymotrypsin. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding (75 vs. 70 degrees C; 1.52 vs. 1.05 M).     

Initiation and synthesis of the Streptococcus pneumoniae type 3 capsule on a phosphatidylglycerol membrane anchor

The type 3 synthase from Streptococcus pneumoniae is a processive beta-glycosyltransferase that assembles the type 3 polysaccharide [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->] by a multicatalytic process. Polymer synthesis occurs via alternate additions of Glc and GlcUA onto the nonreducing end of the growing polysaccharide chain. In the presence of a single nucleotide sugar substrate, the type 3 synthase ejects its nascent polymer and also adds a single sugar onto a lipid acceptor. Following single sugar incorporation from either UDP-[(14)C]Glc or UDP-[(14)C]GlcUA, we found that phospholipase D digestion of the Glc-labeled lipid yielded a product larger than a monosaccharide, while digestion of the GlcUA-labeled lipid resulted in a product larger than a disaccharide. These data indicated that the lipid acceptor contained a headgroup and that the order of addition to the lipid acceptor was Glc followed by GlcUA. Higher-molecular-weight product synthesized in vitro was also sensitive to phospholipase D digestion, suggesting that the same lipid acceptor was being used for single sugar additions and for polymer formation. Mass spectral analysis of the anionic lipids of a type 3 S. pneumoniae strain demonstrated the presence of glycosylated phosphatidylglycerol. This lipid was also observed in Escherichia coli strains expressing the recombinant type 3 synthase. The presence of the lipid primer in S. pneumoniae membranes explained both the ability of the synthase to reinitiate polysaccharide synthesis following ejection of its nascent chain and the association of newly synthesized polymer with the membrane. Unlike most S. pneumoniae capsular polysaccharides, the type 3 capsule is not covalently linked to the cell wall. The present data indicate that phosphatidylglycerol may anchor the type 3 polysaccharide to the cell membrane.     

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).     

Beta-bungarotoxin. Preparation and characterization of crystals suitable for structural analysis

Phospholipases in some snake venoms are potent neurotoxins that target their enzymatic action to the synaptic membrane. One of these is the heterodimeric neurotoxin, beta-bungarotoxin, which binds with a protease inhibitor-like subunit to a presynaptic potassium channel and then blocks neurotransmission with a second subunit that has phospholipase A2 activity. We have prepared and characterized well ordered crystals of the most toxic beta-bungarotoxin isoform, beta 1-bungarotoxin. The crystals are monoclinic, space group C2, with unit cell parameters: a = 176.5 A, b = 39.3 A, c = 92.7 A, and beta = 114.8 degrees. Rotation-function analysis of the Patterson function, as calculated from a 2.3-A data set, reveals an asymmetric unit composed of four heterodimers. These heterodimers appear to be associated as two crystallographically distinct (AB)4 tetramers, each having dihedral D2 symmetry. The two are positioned with equivalent molecular 2-fold axes coincident with crystallographic dyads, but rotated by 55 degrees relative to one another. X-ray analysis of these crystals will permit direct visualization of the specific structural motifs and chemical features that underlie phospholipase neurotoxicity.     

Two-dimensional crystallization of Escherichia coli lactose permease

A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens. Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5.4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation. Depending on reconstitution conditions, trigonal and rectangular crystallographic packing arrangements of these elongated particles assembled into trimers are observed. The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection maps calculated to a resolution of 2 nm show that these crystals consist of two layers.     

Crystallization and preliminary X-ray analysis of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae

A truncated variant of TPK1, the yeast cAMP-dependent protein kinase catalytic subunit, was overexpressed in an engineered strain of Saccharomyces cerevisiae, purified by liquid chromatography, and crystallized from solutions of 2-propanol and magnesium at alkaline pH. The crystals are hexagonal dipyramids, space group P6(1)22 (P6(5)22), with unit-cell parameters a = b = 61 A, c = 320 A. Large single crystals suitable for diffraction analysis are obtainable by microseeding, and diffract beyond 2.8-A resolution. Crystal density measurements reveal 12 kinase monomers per unit cell with a single kinase monomer per asymmetric unit.