Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis-trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis-trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the β-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively

The N-terminal Segment of Recombinant Porcine Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation of Catalysis

Residues 1–10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reducesk cat by 30-fold and Mg2+ affinity by 20-fold and eliminates cooperativity in Mg2+ activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mM. Deletion of the first seven residues reduces k cat and Mg2+ affinity significantly but has no effect on AMP inhibition. The mutation of Asp9 to alanine reproduces the weakened affinity for Mg2+ observed in the deletion mutants, and the mutation of Ile10 to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52–72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxin-regulated FBPase from the chloroplast

Crystal structure and evolution of a prokaryotic glucoamylase

The first crystal structures of a two-domain, prokaryotic glucoamylase were determined to high resolution from the clostridial species Thermoanaerobacterium thermosaccharolyticum with and without acarbose. The N-terminal domain has 18 antiparallel strands arranged in β-sheets of a super-β-sandwich. The C-terminal domain is an (α/α)6 barrel, lacking the peripheral subdomain of eukaryotic glucoamylases. Interdomain contacts are common to all prokaryotic Family GH15 proteins. Domains similar to those of prokaryotic glucoamylases in maltose phosphorylases (Family GH65) and glycoaminoglycan lyases (Family PL8) suggest evolution from a common ancestor. Eukaryotic glucoamylases may have evolved from prokaryotic glucoamylases by the substitution of the N-terminal domain with the peripheral subdomain and by the addition of a starch-binding domain

Mechanism of Action of Escherichia coli Phosphoribosylaminoimidazolesuccinocarboxamide Synthetase

The conversion of ATP, l-aspartate, and 5-aminoimidazole-4-carboxyribonucleotide (CAIR) to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP, and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represents the eighth step of de novo purine nucleotide biosynthesis. SAICAR synthetase and other enzymes of purine biosynthesis are targets of natural products that impair cell growth. Prior to this study, no kinetic mechanism was known for any SAICAR synthetase. Here, a rapid equilibrium random ter-ter kinetic mechanism is established for the synthetase from Escherichia coli by initial velocity kinetics and patterns of linear inhibition by IMP, adenosine 5‘-(β,γ-imido)triphosphate (AMP-PNP), and maleate. Substrates exhibit mutual binding antagonism, with the strongest antagonism between CAIR and either ATP or l-aspartate. CAIR binds to the free enzyme up to 200-fold more tightly than to the ternary enzyme−ATP−aspartate complex, but the latter complex may be the dominant form of SAICAR synthetase in vivo. IMP is a competitive inhibitor with respect to CAIR, suggesting the possibility of a hydrogen bond interaction between the 4-carboxyl and 5-amino groups of enzyme-bound CAIR. Of several aspartate analogues tested (hadacidin, l-malate, succinate, fumarate, and maleate), maleate was by far the best inhibitor, competitive with respect to l-aspartate. Inhibition by IMP and maleate is consistent with a chemical mechanism for SAICAR synthetase that parallels that of adenylosuccinate synthetase

Interaction of Tl+ with Product Complexes of Fructose-1,6-bisphosphatase

The dissociation equilibrium constant for heparin binding to antithrombin III (ATIII) is a measure of the cofactor\u27s binding to and activation of the proteinase inhibitor, and its salt dependence indicates that ionic and non-ionic interactions contribute ∼40 and ∼60% of the binding free energy, respectively. We now report that phenylalanines 121 and 122 (Phe-121 and Phe-122) together contribute 43% of the total binding free energy and 77% of the energy of non-ionic binding interactions. The large contribution of these hydrophobic residues to the binding energy is mediated not by direct interactions with heparin, but indirectly, through contacts between their phenyl rings and the non-polar stems of positively charged heparin binding residues, whose terminal amino and guanidinium groups are thereby organized to form extensive and specific ionic and non-ionic contacts with the pentasaccharide. Investigation of the kinetics of heparin binding demonstrated that Phe-122 is critical for promoting a normal rate of conformational change and stabilizing AT*H, the high affinity-activated binary complex. Kinetic and structural considerations suggest that Phe-122 and Lys-114 act cooperatively through non-ionic interactions to promote P-helix formation and ATIII binding to the pentasaccharide. In summary, although hydrophobic residues Phe-122 and Phe-121 make minimal contact with the pentasaccharide, they play a critical role in heparin binding and activation of antithrombin by coordinating the P-helix-mediated conformational change and organizing an extensive network of ionic and non-ionic interactions between positively charged heparin binding site residues and the cofactor

Mutations in the Hinge of a Dynamic Loop Broadly Influence Functional Properties of Fructose-1,6-bisphosphatase

Loop 52–72 of porcine fructose-1,6-bisphosphatase may play a central role in the mechanism of catalysis and allosteric inhibition by AMP. The loop pivots between different conformational states about a hinge located at residues 50 and 51. The insertion of proline separately at positions 50 and 51 reduces k cat by up to 3-fold, with no effect on the K m for fructose 1,6-bisphosphate. TheK a for Mg2+ in the Lys50→ Pro mutant increases ∼15-fold, whereas that for the Ala51 → Pro mutant is unchanged. Although these mutants retain wild-type binding affinity for AMP and the fluorescent AMP analog 2′(3′)-O-(trinitrophenyl)adenosine 5′-monophosphate, the K i for AMP increases 8000- and 280-fold in the position 50 and 51 mutants, respectively. In fact, the mutation Lys50 → Pro changes the mechanism of AMP inhibition with respect to Mg2+ from competitive to noncompetitive and abolishes K+ activation. The K i for fructose 2,6-bisphosphate increases ∼20- and 30-fold in the Lys50 → Pro and Ala51 → Pro mutants, respectively. Fluorescence from a tryptophan introduced by the mutation of Tyr57suggests an altered conformational state for Loop 52–72 due to the proline at position 50. Evidently, the Pro50 mutant binds AMP with high affinity at the allosteric site, but the mechanism of allosteric regulation of catalysis has been disabled

Crystallization and preliminary X-ray analysis of the Plasmodium falciparum apicoplast DNA polymerase

Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals of P. falciparum apPOL are reported. Data complete to 3.5 Å resolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space group P6522 (unit-cell parameters a = b = 141.8, c = 149.7 Å, α = β = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress