Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase.

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.

Time-resolved ligand exchange reactions: kinetic models for competitive inhibitors with recombinant human renin.

The on and off rate constants (kon and koff) were determined for a series of peptidomimetic, competitive inhibitors of human renin using a novel binding assay. The method entails analyzing a pair of ligand exchange reactions in which a dansylated inhibitor serves as the fluorescent probe. The first in the pair of reactions involves preincubating renin with the probe and initiating the reaction by addition of a sample inhibitor; the second reaction involves preincubating renin with the sample inhibitor and initiating the reaction by addition of probe. Both reactions yield progress curves which contain complementary information concerning the kon and koff of each ligand. The two curves are fitted simultaneously using models derived from the differential rate equations describing the ligand exchange process. The kon and koff rate constants for the probe were 6.85 x 10(6) M-1 s-1 and 2.96 x 10(-4) s-1, respectively, giving a calculated Kd of 43.2 pM. The Kd values for the inhibitor series varied over 2 orders of magnitude (27-2320 pM), while the individual kon (10(6)-10(7) M-1 s-1) and koff (10(-4)-10(-3) s-1) constants varied only over 1 order of magnitude.

Effect of deoxycoformycin and Val-boroPro on the associated catalytic activities of lymphocyte CD26 and ecto-adenosine deaminase.

CD26 and ecto-adenosine deaminase (ADA) are found associated on the plasma membrane of T lymphocytes and each possess distinct catalytic activities. CD26 has a proteolytic activity identical to dipeptidylpeptidase IV (DPPIV; E.C. 3.4.14.5), and ecto-ADA (E.C. 3.5.4.4) degrades extracellular adenosine. The cell surface expression of CD26 and ecto-adenosine deaminase (ecto-ADA) is regulated on stimulated T lymphocytes, and ADA binding to CD26 produces a synergistic costimulatory response with T cell receptor activation. This study addresses the potential regulation by allosteric interactions of the catalytic activities of CD26 associated with ecto-ADA, which could define the mechanism of the synergism observed in T cell signaling. Cell lines genetically deficient in ADA, ligands for ADA such as adenosine, and a specific inhibitor of ADA, deoxycoformycin, were used to define the effect of ADA activity on CD26 DPPIV activity and affinity for dipeptide substrate. Conversely, a recombinant Chinese hamster ovary cell line expressing human CD26 with or without a mutation in the DPPIV catalytic domain, and the boronic acid inhibitor Val-boroPro, were used to determine the effect of DPPIV activity on ecto-ADA activity and association with CD26. These studies found no significant allosteric interaction between the catalytic activities of CD26 and ecto-ADA when associated. Therefore, signaling events in T cells involving costimulation with CD26 and ecto-ADA and the synergism observed upon ADA binding to CD26 occur independently of the catalytic activities of these cell surface molecules.

Identification and characterization of the N-ethylmaleimide-sensitive site in lambda-integrase.

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase. Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected. In this report, the NEM-sensitive site was identified by modifying Int with [14C]NEM. Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of [14C]NEM incorporation was subjected to amino acid sequencing. The results indicate that the primary site of [14C]NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine. The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment. The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to form the arm-type recognition pocket. The loss of arm-type DNA binding and the concomitant loss of recombination function as a result of NEM modification must be due to the presence of the maleimide moiety and not due to loss of a critical cysteine contact.

Suicide recombination substrates yield covalent lambda integrase-DNA complexes and lead to identification of the active site tyrosine.

High levels of covalent integrase-DNA complexes accumulate when suicide substrates containing a medial nick within the overlap region are nicked by lambda integrase protein. The tyrosine residue at position 342 is shown to form a covalent bond with DNA at the sites of strand exchange. A mutant integrase in which this tyrosine is changed to phenylalanine is devoid of both topoisomerase and recombinase activity but still binds to both core- and arm-type DNA binding sites with an affinity comparable to wild-type integrase. Tyrosine-342 is located within a 40-amino acid region that is conserved among 15 known recombinases comprising the "integrase family." The present results show that this small region of homology participates in catalysis of strand transfer.

Inhibition of dipeptidyl peptidase IV (CD26) by peptide boronic acid dipeptides.

Peptide boronic acid dipeptide compounds were analyzed for their ability to inhibit recombinant human dipeptidylpeptidase IV (CD26, DPPIV). Rate constants for the peptide boronates are difficult to obtain because the active boronic acid dipeptide exists in equilibrium with a cyclic inactive species in aqueous solution. Rate constants were determined for the inhibition of DPPIV using several peptide boronates at different pH values. Val-boroPro forms the most tightly bound complex with DPPIV; the first order half life for dissociation of the inactive enzyme-inhibitor complex at 23 degrees C is approximately 27 days.

Kinetic characterization of human immunodeficiency virus type 1 protease: determination of inhibitor rate constants during dynamic monomer-dimer interconversion.

A numerical method was applied to a system of differential rate equations describing the monomer-dimer-inhibitor (M-D-I) interaction involving human immunodeficiency virus type 1 protease and a peptidomimetic, competitive inhibitor. Two pairs of progress curves were obtained, one involving the M-D interaction and the other the M-D-I interaction. Each pair of reactions was designed to begin with extreme conditions and end at the identical equilibrium position. The results were compared with analytical (exact mathematical) methods reported previously. Good agreement between the two methods was observed at high- and low-salt conditions for the rates of monomer association and dimer dissociation. Not surprisingly, however, the major difference was observed in the analyses involving the M-D-I interaction, since analytical methods cannot account for dimer dissociation in the presence of inhibitor. While the estimates for the inhibitor off rate were comparable for high-salt conditions (where dimer dissociation is minimized), the analytical method underestimated this parameter for low-salt conditions by an order of magnitude, the consequence of mistaking inactive M for inactive DI.

Autonomous DNA binding domains of lambda integrase recognize two different sequence families.

The 40 kd lambda Integrase protein is shown to contain two autonomous DNA binding domains with different sequence specificities. Competition experiments in which the binding activity of Int is assayed through nuclease protection demonstrate the functional independence of the two DNA recognition specificities. Proteolytic cleavage of Int and footprinting analysis of the resulting two major peptides allow the physical separation and identification of two DNA binding domains: an amino-terminal peptide that interacts with "arm-type" sites and a carboxy-terminal peptide that binds to "core-type" sequences. In addition, the data suggest that the two domains can bind DNA simultaneously, consistent with a model in which Integrase would link two disparate DNA sequences.

Determination of kinetic rate constants for the binding of inhibitors to HIV-1 protease and for the association and dissociation of active homodimer.

Association and dissociation rate constants for a competitive inhibitor of HIV-1 protease were determined by a novel method employing a pair of integrated rate equations. This method, termed the paired progress curve method, is both rapid and reproducible. Progress curves, taken at a single concentration of inhibitor, are analyzed simultaneously to determine association and dissociation rate constants, the concentration of active sites, and the catalytic rate constant. The method is applied to BILA 398, a compound for which the cocrystal structure with HIV-2 protease has been reported recently [Tong, L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8387-8391]. This compound exhibited an association constant of 1.6 x 10(7) M-1 s-1 and a dissociation constant of 1.0 x 10(-4) s-1 corresponding to a binding affinity constant of 6.4 x 10(-12) M. During the course of the analysis, nonlinearity was observed in control reactions containing enzyme and substrate only. This was subsequently shown to be due to a reversible inactivation process resulting from enzyme dilution. Integrated rate equations were developed on the basis of the dissociation of active dimeric enzyme during dilution and a reassociation of dilute monomers following the addition of substrate. The equations were modeled to the data, yielding a dissociation constant of 1.9 x 10(-3) s-1 and an association constant of 9.2 x 10(5) M-1 s-1 for the monomer-dimer interconversion process. This corresponds to an equilibrium constant of 4 x 10(-9) M for the dimerization of HIV-1 protease.

A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket.

The crystal structure of human p38 mitogen-activated protein (MAP) kinase in complex with a potent and highly specific pyridinyl-imidazole inhibitor has been determined at 2.0 A resolution. The structure of the kinase, which is in its unphosphorylated state, is similar to that of the closely-related ERK2. The inhibitor molecule is bound in the ATP pocket. A hydrogen bond is made between the pyridyl nitrogen of the inhibitor and the main chain amido nitrogen of residue 109, analogous to the interaction from the N1 atom of ATP. The crystal structure provides possible explanations for the specificity of this class of inhibitors. Other protein kinase inhibitors may achieve their specificity through a similar mechanism. The structure also reveals a possible second binding site for this inhibitor, with currently unknown function.