Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation ...loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine → phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.
The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein ...crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.5 A, and c = 138.9 A. There is one 156,000 Da protein tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 86% complete data from 10.0 to 2.8 A to a crystallographic Rfactor of 0.198. Density modification by noncrystallographic symmetry averaging was used during model building. The final model of the L. mexicana GAPDH tetramer shows small deviations of less than 0.5 degrees from ideal 222 molecular symmetry. The structure of L. mexicana GAPDH is very similar to that of glycosomal GAPDH from the related trypanosomatid Trypanosoma brucei. A significant structural difference between L. mexicana GAPDH and most previously determined GAPDH structures occurs in a loop region located at the active site. This unusual loop conformation in L. mexicana GAPDH occludes the inorganic phosphate binding site which has been seen in previous GAPDH structures. A new inorganic phosphate position is observed in the L. mexicana GAPDH structure. Model building studies indicate that this new anion binding site is well situated for nucleophilic attack of the inorganic phosphate on the thioester intermediate in the GAPDH-catalyzed reaction. Since crystals of L. mexicana GAPDH can be grown reproducibly and diffract much better than those of T. brucei GAPDH, L. mexicana GAPDH will be used as a basis for structure-based drug design targeted against trypanosomatid GAPDHs.
Background:
Escherichia coli heat-labile enterotoxin (LT) is the causative agent of traveller's diarrhoea, and it is also responsible for the deaths of hundreds of thousands of children per year in ...developing countries. LT is highly homologous in sequence, structure and function to cholera toxin (CT). Both toxins attack intestinal epithelial cells via specific binding to the branched pentasaccharide of ganglioside G
M1 at the cell surface. A receptor-binding antagonist which blocked this interaction would potentially constitute a prophylactic drug conferring protection both against the severe effects of cholera itself and against the milder but more common disease caused by LT.
Results: Four derivatives of the simple sugar galactose, members of a larger series of receptor antagonists identified by computer modeling and competitive binding studies, have been co-crystallized with either the full LT AB
5 holotoxin or the LT B pentamer. These crystal structures have provided detailed views of the toxin in complex with each of the four antagonists: melibionic acid at 2.8 Å resolution, lactulose at 2.65 Å resolution, metanitrophenylgalactoside (MNPG) at 2.2 Å resolution and thiodigalactoside (TDG) at 1.7 Å resolution. The binding mode of each galactose derivative was observed 5–15 times, depending on the number of crystallographically independent toxin B pentamers per asymmetric unit. There is a remarkable consistency, with one important exception, in the location and hydrogen-bonding involvement of well-ordered water molecules at the receptor-binding site.
Conclusions: The bound conformations of these receptor antagonist compounds preserve the toxin–galactose interactions previously observed for toxin–sugar complexes, but gain additional favorable interactions. The highest affinity compound, MNPG, is notable in that it displaces a water molecule that is observed to be well-ordered in all other previous and current crystal structures of toxin–sugar complexes. This could be a favorable entropic factor contributing to the increased affinity. The highest affinity members of the present set of antagonists (MNPG and TDG) bury roughly half (400 Å
2) of the binding-site surface covered by the full receptor G
M1 pentasaccharide, despite being considerably smaller. This provides an encouraging basis for the creation of subsequent generations of derived compounds that can compete effectively with the natural receptor.
αB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation ...x-ray solution scattering study of a truncation mutant from the human αB-crystallin (αB57–157), a dimeric protein that comprises the α-crystallin domain of the αB-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the α-crystallin domain should be close to that of the small heat-shock protein from Methanococcusjannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the α-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the α-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite β-sheet. This model agrees with the recent spin labeling results and suggests that the αB-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of αB-crystallin complexes of variable sizes and compositions.
αB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly
and can act as a molecular chaperone. We report a synchrotron radiation ...x-ray solution scattering study of a truncation mutant
from the human αB-crystallin (αB57â157), a dimeric protein that comprises the α-crystallin domain of the αB-crystallin and
retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric
fold of the α-crystallin domain should be close to that of the small heat-shock protein from Methanococcus
jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates
significantly from the experimental scattering by the α-crystallin domain, pointing to different quaternary structures of
the two proteins. A rigid body modeling against the solution scattering data yields a model of the α-crystallin domain revealing
a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which
form a four-stranded, antiparallel, intersubunit composite β-sheet. This model agrees with the recent spin labeling results
and suggests that the αB-crystallin is composed by flexible building units with an extended surface area. This flexibility
may be important for biological activity and for the formation of αB-crystallin complexes of variable sizes and compositions.
L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad substrate specificity and utilizes a wide range of 2-oxo acids branched at the C4 atom. Modifications have been made to the ...sequence of the NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and alter the region of substrate specificity towards various 2-oxocarbonic acids. All variations were based on a 3D-structure model of the enzyme using the X-ray coordinates of the functionally related L-lactate dehydrogenase (L-LDH) from dogfish as a template. This protein displays only 23% sequence identity to L-HicDH. The active site of L-HicDH was modelled by homology to the L-LDH based on the conservation of catalytically essential residues. Substitutions of the active site residues Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify their unique participation in substrate recognition and orientation. The kinetic properties of the L239A, L239M, L236V and T245A enzyme variants confirmed the structural model of the active site of L-HicDH. The substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate, phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the active site of the subsequently refined model. In order to design dehydrogenases with an improved substrate specificity towards keto acids branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236 and Thr245 were introduced and resulted in mutant enzymes with completely different substrate specificities. The substitution T245A resulted in a relative shift of substrate specificity for keto-tert-leucine of more than 17000 compared with the 2-oxocaproate (kcat/KM). For the substrates branched at C4 a relative shift of up to 500 was obtained for several enzyme variants. A total of nine mutations were introduced and the kinetic data for the set of six substrates were determined for each of the resulting mutant enzymes. These were compared with those of the wild-type enzyme and rationalized by the active site model of L-HicDH. An analysis of the enzyme variants provided new insight into the residues involved in substrate binding and residues of importance for the differences between LDHs and HicDH. After the protein design project was complete the X-ray structure of the enzyme was solved in our group. A comparison between the model and the experimental 3D structure proved the quality of the model. All the variants were designed, expressed and tested before the 3D structure became available.
Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one ...240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.
Members of the cholera toxin family, including Escherichia coli heat-labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from ...NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT-II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT-I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.
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