Markus Grütter

Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone.

The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity. The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 Å. The asymmetric unit contains a (p17/p12)2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography. The overall topology of CPP32 is very similar to that of interleukin-1β-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32. A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site. In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor. The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE. This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition. This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.

Refined crystal structures of subtilisin Novo in complex with wild-type and two mutant eglins: Comparison with other serine proteinase inhibitor complexes

The crystal structures of the complexes formed between subtilisin Novo and three inhibitors, eglin c, Arg45-eglin c and Lys53-eglin c have been determined using molecular replacement and difference Fourier techniques and refined at 2.4 A, 2.1 A, and 2.4 A resolution, respectively. The mutants Arg45-eglin c and Lys53-eglin c were constructed by site-directed mutagenesis in order to investigate the inhibitory specificity and stability of eglin c. Arg45-eglin became a potent trypsin inhibitor, in contrast to native eglin, which is an elastase inhibitor. This specificity change was rationalized by comparing the structures of Arg45-eglin and basic pancreatic trypsin inhibitor and their interactions with trypsin. The residue Arg53, which participates in a complex network of hydrogen bonds formed between the core and the binding loop of eglin c, was replaced with the shorter basic amino acid lysine in the mutant Lys53-eglin. Two hydrogen bonds with Thr44, located in the binding loop, can no longer be formed but are partially restored by a water molecule bound in the vicinity of Lys53. Eglin c in complexes with both subtilisin Novo and subtilisin Carlsberg was crystallized in two different space groups. Comparison of the complexes showed a rigid body rotation for the eglin c core of 11.5 degrees with respect to the enzyme, probably caused by different intermolecular contacts in both crystal forms.

A new structural class of serine protease inhibitors revealed by the structure of the hirustasin–kallikrein complex

BACKGROUND Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. RESULTS The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. CONCLUSIONS The crystal structure of the kallikrein-hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel beta sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

Crystallization and structure solution of p53 (residues 326-356) by molecular replacement using an NMR model as template.

The molecular replacement method is a powerful technique for crystal structure solution but the use of NMR structures as templates often causes problems. In this work the NMR structure of the p53 tetramerization domain has been used to solve the crystal structure by molecular replacement. Since the rotation- and translation-functions were not sufficiently clear, additional information about the symmetry of the crystal and the protein complex was used to identify correct solutions. The three-dimensional structure of residues 326-356 was subsequently refined to a final R factor of 19.1% at 1.5 A resolution.

Comparative analysis of the X-ray structures of HIV-1 and HIV-2 proteases in complex with CGP 53820, a novel pseudosymmetric inhibitor

BACKGROUND The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Two subtypes of the virus, HIV-1 and HIV-2, have been characterized. The protease enzymes from these two subtypes, which are aspartic acid proteases and have been found to be essential for maturation of the infectious particle, share about 50% sequence identity. Differences in substrate and inhibitor binding between these enzymes have been previously reported. RESULTS We report the X-ray crystal structures of both HIV-1 and HIV-2 proteases each in complex with the pseudosymmetric inhibitor, CGP 53820, to 2.2 A and 2.3 A, respectively. In both structures, the entire enzyme and inhibitor could be located. The structures confirmed earlier modeling studies. Differences between the CGP 53820 inhibitory binding constants for the two enzymes could be correlated with structural differences. CONCLUSIONS Minor sequence changes in subsites at the active site can explain some of the observed differences in substrate and inhibitor binding between the two enzymes. The information gained from this investigation may help in the design of equipotent HIV-1/HIV-2 protease inhibitors.

Crystallization and preliminary X-ray analysis of recombinant human transforming growth factor β2

Recombinant human transforming growth factor β2 (TGF‐β2) was cloned and expressed in E. coli. The protein was isolated from inclusion bodies, renatured and purified to a single component as judged by reversed‐phase HPLC. The recombinant TGF‐β2 was shown to have a biological activity equal to that of native TGF‐β2 in a fibroblast migration assay. Pure, active recombinant TGF‐β2 has been crystallized from polyethylene glycol 400. The trigonal crystals of spacegroup P3121 or P3221 have unit cell dimensions of a=b=60.6 Å, c=75.2 Å and diffract beyond 2.0 Å.

The structure of murine interleukin-1 beta at 2.8 A resolution.

The three-dimensional structure of recombinant murine interleukin-1 beta has been solved by X-ray crystallographic techniques to 2.8 A resolution and refined to a crystallographic R factor of 0.192. Although murine interleukin-1 beta crystallizes in the same space group as human interleukin-1 beta with almost identical unit cell dimensions, the packing of the molecules is quite different. The murine interleukin-1 beta structure was solved by molecular replacement using the refined structure of human interleukin-1 beta as trial structure, and found to be related to the human structure by a nearly perfect twofold rotation about the crystallographic y-axis and a 14 degrees rotation about the z-axis, with no translation. The folding of murine interleukin-1 beta is similar to that found for the human variant, consisting of 12 beta strands wrapped around a core of hydrophobic side chains in a tetrahedron-like fashion. Significant differences with respect to the human structure are seen at the N terminus and in 4 of the 11 loops connecting the 12 beta strands.

X-ray crystal structure of the serine proteinase inhibitor eglin c at 1.95 Å resolution

The crystal structure eglin c. naturally occurring in the leech Hirudo medicinalis, is known from its complexes with various serine proteinases, but the crystallization of free eglin c has not yet been reported. A method is described for growing well‐diffracing crystals of free eglic c from highly concentrated protein solutions (≈2OO mg/ml). The space group of the orthorhombic crystals was determined to be P212121 with unit cell parameters a = 32.6. b = 42.0. c = 44.1 Å. The structure or free eglin c was resolved at 1.95 Å resolution by Patterson search methods. The final model contains all 70 amino acids of eglin c and 125 water molecules. In comparison to the eglin structure known from its complexes with proteinases, only small differences have been observed in free eglin c. However, the reactive site‐binding loop and a few residues on the surface of eglin have been found in different conformations due to crystal contacts. In contrast to the complex structures, the first seven amino acids of the highly flexible amino terminus can be located. Crystallographic refinement comprised molecular dynamics refinement, classical retrained least‐squares refinement and individual isotropic atomic temperature refinement. The final R‐factor is 15.8%.

Proteinase inhibitors: another new fold.

The three-dimensional descriptions of two serine proteinase inhibitors from the parasitic worm Ascaris--one in solution and the other in a complex with its substrate--reveal a new structural motif.

Refolding, isolation and characterization of crystallizable human interferon-α8 expressed in Saccharomyces cerevisiae

Human interferon-α8 was expressed in Saccharomyces cerevisiae and found to accumulate intracellularly in an insoluble form. The protein could be solubilized and converted to a biologically active form with high yield by a denaturation-refolding procedure. The interferon-α8 was further purified to apparent homogeneity by copper-chelate affinity chromatography and anion-exchange chromatography and fully characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, mass spectrometry, circular-dichroism (CD) spectroscopy and specific activity. Secondary-structure predictions from CD spectroscopy indicate that the molecule is correctly folded. Peptide mapping supported the correct sequence and the expected disulfide-bridge connectivity. The purified protein elutes on reversed-phase high-pressure liquid chromatography (RP-HPLC) as two peaks. Electrospray mass spectrometry and N-terminal sequence analysis of the minor component indicated the existence of an N-terminal acetyl group for the later eluting HPLC-component. In anti-viral assays, the two IFN forms were equally active. Hexagonal crystals of this interferon preparation could be obtained. On the basis of the electrophoretic mobility, HPLC profile, and biological activity assay, the crystalline material was judged to be identical to the uncrystallized interferon. Interferon in crystallized form was found to be stable for up to 24 months and, therefore, could be used for long-term storage, particularly for material intended for clinical use.