The amino-terminal residues in the crystal structure of connective tissue activating peptide-III (des10) block the ELR chemotactic sequence.

alpha-Chemokines comprise a family of cytokines that are chemotactic for neutrophils and have a structure similar to platelet factor 4 (PF4), in which the first two cysteine residues are separated by one residue (Cys-X-Cys). The two alpha-chemokines, connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2 (NAP-2), are carboxyl-terminal fragments of platelet basic protein (PBP) that are generated by monocyte-derived proteases. NAP-2 strongly stimulates neutrophils that are present during inflammation whereas its precursors, PBP and CTAP-III, are inactive, although they also possess the highly conserved, amino-terminal sequence, Glu-Leu-Arg (ELR), that is critical for receptor binding. To resolve this conundrum, we have determined the crystal structure of recombinant Asp-CTAP, which has ten fewer amino-terminal residues than CTAP-III but five more than NAP-2. The space group is P2(1)with unit cell dimensions a = 43.8 A, b = 76.8 A, c = 43.8 A, and beta =97.0 degrees, and a tetramer in the asymmetric unit. The molecular replacement method, with the NAP-2 tetramer as a starting model, was used to determine the initial phase information. The final R-factor is 0.196 (Rfree = 0.251) for 2sigma data from 7.0 to 1.75 A resolution. This high-resolution model of Asp-CTAP is the longest defined structure of an alpha-chemokine to date. The electron density map shows an over-all structure for Asp-CTAP that is very similar to that of NAP-2, but with the additional five amino-terminal residues folding back through a type-II turn, thereby stabilizing the oligomeric "inactive" state, and masking the critical ELR receptor binding region that is exposed in the structure of NAP-2.

The crystal structure of recombinant human neutrophil-activating peptide-2 (M6L) at 1.9-A resolution.

Neutrophil-activating peptide-2 (NAP-2) is a 70-residue carboxyl-terminal fragment of platelet basic protein, which is found in the alpha-granules of human platelets. NAP-2, which belongs to the CXC family of chemokines that includes interleukin-8 and platelet factor 4, binds to the interleukin-8 type II receptor and induces a rise in cytosolic calcium, chemotaxis of neutrophils, and exocytosis. Crystals of recombinant NAP-2 in which the single methionine at position 6 was replaced by leucine to facilitate expression belong to space group P1 (unit cell parameters a = 40.8, b = 43.8, and c = 44.7 A and alpha = 98.4 degrees, beta = 120.3 degrees, and gamma = 92.8 degrees), with 4 molecules of NAP-2 (Mr = 7600) in the asymmetric unit. The molecular replacement solution calculated with bovine platelet factor 4 as the starting model was refined using rigid body refinement, manual fitting in solvent-leveled electron density maps, simulated annealing, and restrained least squares to an R-factor of 0.188 for 2 sigma data between 7.0- and 1.9-A resolution. The final refined crystal structure includes 265 solvent molecules. The overall tertiary structure, which is similar to that of platelet factor 4 and interleukin-8, includes an extended amino-terminal loop, three strands of antiparallel beta-sheet arranged in a Greek key fold, and one alpha-helix at the carboxyl terminus. The Glu-Leu-Arg sequence that is critical for receptor binding is fully defined by electron density and exhibits multiple conformations.

The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.

Expression of synthetic genes encoding fused proteins under tight control of modified regulatory regions of the colicin operon.

A versatile expression vector system for construction of gene and protein fusions, specific radiolabeling of gene products and high-level protein production is described. Expression cassettes were constructed containing structural genes encoding native and analog forms of connective tissue-activating peptide-III (CTAP-III), beta-thromboglobulin, neutrophil-activating protein and modified regulatory sequences derived from the colicin E1 operon. Gene expression was enhanced by changes in the colicin promoter that increased the transcription initiation rate both in vivo and in vitro, and by deletion of a sequence affecting catabolite repression. High-level expression, producing recombinant protein up to 30% of the total cellular protein, was induced rapidly after stimulation of the SOS response by using either mitomycin C or nalidixic acid, by temperature shift using temperature-sensitive mutations in the LexA or RecA proteins, or by UV light. The presence of radiolabeled amino acids during induction resulted in greater than 95% preferential labeling of recombinant proteins. CTAP-III remained stable for more than 6 h following decay of the inducing signal. The use of CTAP-III in protein fusions improved stability of several therapeutically useful proteins including the thrombin-specific inhibitor, hirudin and a cell receptor-binding domain of laminin, when they were produced in Escherichia coli.

Chemical modifications and amino acid substitutions in recombinant hirudin that increase hirudin-thrombin affinity.

Recombinant hirudin (r-hirudin), unlike the naturally occurring leech protein, lacks a sulfate ester on Tyr-63 which reduces its binding affinity to thrombin by 3-10-fold. We demonstrate that nitration or iodination of Tyr-63 restores hirudin-thrombin affinity to levels similar to or exceeding that of the natural inhibitor. In contrast, nitration of Tyr-3 reduces the affinity of hirudin for thrombin. These chemical modifications results in multiple reaction products that are readily separated by reverse-phase HPLC. The mechanism of the observed changes in thrombin affinity may involve a reduction in the pK of the hydroxyl group of tyrosine due to substitution of the electrophilic iodo or nitro group on the phenyl ring, resulting in an increased negative charge at neutral pH. For Tyr-63, this effect mimics the sulfatotyrosine of natural hirudin, leading to an increased thrombin affinity at the anion-binding exosite. For Tyr-3, the increased polarity may destabilize its interaction within the apolar-binding site of thrombin. Substitution of the highly conserved Tyr-3 residue with Phe or Trp not only enables specific and quantitative chemical modification at Tyr-63 but also independently increases hirudin-thrombin affinity. Kinetic analysis of thrombin inhibition showed that enhanced binding by r-hirudin(nitro-Tyr-63) is due to an increase in the association rate between hirudin and thrombin whereas the reduced binding of r-hirudin(nitro-Tyr-3) results from a large increase in the dissociation rate. These observations indicate that specific segments within both the amino- and carboxy-terminal regions of hirudin interact with thrombin.

Hirudin: amino-terminal residues play a major role in the interaction with thrombin.

Amino acid substitutions within the amino-terminal 5 residues of the thrombin-specific inhibitor hirudin dramatically alter its ability to inhibit the thrombin-catalyzed hydrolysis of both a chromogenic substrate and fibrinogen. Replacing the highly conserved Tyr-3 residue with Trp or Phe increases hirudin's affinity for thrombin 3-6-fold (decreases the inhibition constant, Ki) whereas Thr results in a 450-fold increase in Ki. A more extensive modification involving deletion of the amino-terminal Val, and Tyr-3----Val, Thr-4----Gln, and Asp-5----Ile replacement, results in a large reduction in thrombin inhibitory activity corresponding to greater than a 10(7)-fold increase in Ki and a 10(3)-fold increase in IC50, using D-Phe-L-pipecolyl-Arg-p-nitroanilide (S-2238) and fibrinogen, respectively, as substrates. Kinetic analysis of these mutant proteins and synthetic peptide fragments and available structural information on thrombin and hirudin derived from protein crystallography and two-dimensional NMR studies indicate that the amino-terminal region of hirudin binds at the apolar binding/active site region of thrombin, with Tyr-3 occupying the S3 specificity site. The large effect of these modifications on hirudin activity suggests that alteration of the amino-terminal segment can destabilize the interaction of other regions of hirudin with thrombin.

Structure-function and refolding studies of the thrombin-specific inhibitor hirudin.

We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human alpha-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration (greater than 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudin:thrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42-65 and 51-65 [but not (Ala22)-6-28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 microM, respectively, at a thrombin concentration of 1 microM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.

The NIMH Depression Awareness, Recognition, and Treatment Program: structure, aims, and scientific basis.

Unrecognized, untreated, and undertreated depressive disorders extract an inordinate human and economic cost, despite the availability of an extensive array of effective clinical interventions. To enhance the availability and quality of care, the National Institute of Mental Health launched the Depression Awareness, Recognition, and Treatment Program, a multiphase information and education program designed to alert health professionals and the general public to the fact that depressive disorders are common, serious, and treatable. The authors review the development of this program, describing the professional education efforts it supports in anticipation of increased demand for services, the public education campaign launched in May 1988, and highlights of the scientific advances that make the program feasible and timely.