Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology.

The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in "inclusion bodies" (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24 °C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3 × 10(7)IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form.

Flexibility and variability of TIMP binding: X-ray structure of the complex between collagenase-3/MMP-13 and TIMP-2.

The excessive activity of matrix metalloproteinases (MMPs) contributes to pathological processes such as arthritis, tumor growth and metastasis if not balanced by the tissue inhibitors of metalloproteinases (TIMPs). In arthritis, the destruction of fibrillar (type II) collagen is one of the hallmarks, with MMP-1 (collagenase-1) and MMP-13 (collagenase-3) being identified as key players in arthritic cartilage. MMP-13, furthermore, has been found in highly metastatic tumors. We have solved the 2.0 A crystal structure of the complex between the catalytic domain of human MMP-13 (cdMMP-13) and bovine TIMP-2. The overall structure resembles our previously determined MT1-MMP/TIMP-2 complex, in that the wedge-shaped TIMP-2 inserts with its edge into the entire MMP-13 active site cleft. However, the inhibitor is, according to a relative rotation of approximately 20 degrees, oriented differently relative to the proteinase. Upon TIMP binding, the catalytic zinc, the zinc-ligating side chains, the enclosing MMP loop and the S1' wall-forming segment move significantly and in concert relative to the rest of the cognate MMP, and the active site cleft constricts slightly, probably allowing a more favourable interaction between the Cys1(TIMP) alpha-amino group of the inhibitor and the catalytic zinc ion of the enzyme. Thus, this structure supports the view that the central N-terminal TIMP segment essentially defines the relative positioning of the TIMP, while the flanking edge loops determine the relative orientation, depending on the individual target MMP.

Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism.

Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.

A novel strategy for inhibition of alpha-amylases: yellow meal worm alpha-amylase in complex with the Ragi bifunctional inhibitor at 2.5 A resolution.

BACKGROUND: alpha-Amylases catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional alpha-amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and alpha-amylases. The mode of inhibition of alpha-amylases by these cereal inhibitors has so far been unknown. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with RBI was determined at 2.5 A resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3(10)-helical conformation in the complex. RBI's trypsin-binding loop is located opposite the alpha-amylase-binding site, allowing simultaneous binding of alpha-amylase and trypsin. CONCLUSIONS: The binding of RBI to TMA constitutes a new inhibition mechanism for alpha-amylases and should be general for all alpha-amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

Crystal structure of yellow meal worm alpha-amylase at 1.64 A resolution.

The three-dimensional structure of the alpha-amylase from Tenebrio molitor larvae (TMA) has been determined by molecular replacement techniques using diffraction data of a crystal of space group P212121 (a=51.24 A; b=93.46 A; c=96.95 A). The structure has been refined to a crystallographic R-factor of 17.7% for 58,219 independent reflections in the 7.0 to 1.64 A resolution range, with root-mean-square deviations of 0.008 A for bond lengths and 1.482 degrees for bond angles. The final model comprises all 471 residues of TMA, 261 water molecules, one calcium cation and one chloride anion. The electron density confirms that the N-terminal glutamine residue has undergone a post-transitional modification resulting in a stable 5-oxo-proline residue. The X-ray structure of TMA provides the first three-dimensional model of an insect alpha-amylase. The monomeric enzyme exhibits an elongated shape approximately 75 Ax46 Ax40 A and consists of three distinct domains, in line with models for alpha-amylases from microbial, plant and mammalian origin. However, the structure of TMA reflects in the substrate and inhibitor binding region a remarkable difference from mammalian alpha-amylases: the lack of a highly flexible, glycine-rich loop, which has been proposed to be involved in a "trap-release" mechanism of substrate hydrolysis by mammalian alpha-amylases. The structural differences between alpha-amylases of various origins might explain the specificity of inhibitors directed exclusively against insect alpha-amylases.

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure.

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.

Efficient catalysis of disulfide formation during protein folding with a single active-site cysteine.

Protein disulfide isomerases (PDIs) catalyze disulfide bond formation during protein folding in vivo and are essential for viability in eukaryotic cells. They share the active-site sequence C-X-X-C that forms a catalytic disulfide. The recent finding that the EUG1 protein, a PDI-related yeast protein, with C-X-X-S sequence at its active sites can complement PDI-deficiency raised the general question of whether disulfide-isomerase activity is essential for cell viability or whether PDI variants with single active-site thiol groups can be catalytically active as disulfide isomerases. We investigated the function of the catalytic cysteine residues in DsbA, a PDI-related protein required for disulfide formation in the periplasmic space of Escherichia coli, by replacing C30 and C33 with alanine. While the mutant C30A and the double mutant CC30/33AA are inactive, C33A catalyzes disulfide-interchange reactions and oxidative renaturation of the reduced, unfolded thrombin inhibitor hirudin with close to wild-type efficiency. Thus, the single active-site thiol group of C30 is sufficient for disulfide-isomerase activity of the DsbA protein.

Crystal structure of the catalytic domain of MMP-16/MT3-MMP: characterization of MT-MMP specific features.

Membrane-type matrix metalloproteinases (MT-MMPs) have attracted strong attention, because four of them can activate a key player in the tumor scenario, proMMP-2/progelatinase A. In addition to this indirect effect on the cellular environment, these MT-MMPs degrade extracellular matrix proteins, and their overproduction is associated with tumor growth. We have solved the structure of the catalytic domain (cd) of MT3-MMP/MMP-16 in complex with the hydroxamic acid inhibitor batimastat. CdMT3-MMP exhibits a classical MMP-fold with similarity to MT1-MMP. Nevertheless, it also shows unique properties such as a modified MT-specific loop and a closed S1' specificity pocket, which might help to design specific inhibitors. Some MT-MMP-specific features, derived from the crystal structures of MT-1-MMP determined previously and MT3-MMP, and revealed in recent mutagenesis experiments, explain the impaired interaction of the MT-MMPs with TIMP-1. Docking experiments with proMMP-2 show some exposed loops including the MT-loop of cdMT3-MMP involved in the interaction with the proMMP-2 prodomain in the activation encounter complex. This model might help to understand the experimentally proven importance of the MT-loop for the activation of proMMP-2.

Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 A resolution.

The three-dimensional structure of human tissue inhibitor of metalloproteinases-2 (TIMP-2) was determined by X-ray crystallography to 2.1 A resolution. The structure of the inhibitor consists of two domains. The N-terminal domain (residues 1-110) is folded into a beta-barrel, similar to the oligonucleotide/oligosaccharide binding fold otherwise found in certain DNA-binding proteins. The C-terminal domain (residues 111-194) contains a parallel stranded beta-hairpin plus a beta-loop-beta motif. Comparison of the structure of uncomplexed human TIMP-2 with that of bovine TIMP-2 bound to the catalytic domain of human MMP-14 suggests an internal rotation between the two domains of approximately 13 degrees upon binding to the protease. Furthermore, local conformational differences in the two structures that might be induced by formation of the protease-inhibitor complex have been found. The most prominent of these involves residues 27-40 of the A-B beta-hairpin loop. Structure-based alignment of amino acid sequences of representatives of the TIMP family maps the sequence differences mainly to loop regions, and some of these differences are proposed to be responsible for the particular properties of the various TIMP species.

RBI, a one-domain alpha-amylase/trypsin inhibitor with completely independent binding sites.

The bifunctional inhibitor from Ragi (Eleusine coracana Gaertneri) (RBI) is the only member of the alpha-amylase/trypsin inhibitor family that inhibits both trypsin and alpha-amylase. Here, we show that both enzymes simultaneously and independently bind to RBI. The recently solved three-dimensional NMR structure of RBI has revealed that the inhibitor possesses a hitherto unknown fold for serine proteinase and alpha-amylase inhibitors. Despite its different fold, RBI obeys the standard mechanism observed for most protein inhibitors of serine proteinases and is a strong, competitive inhibitor of bovine trypsin (Ki = 1.2 +/- 0.2 nM). RBI is also a competitive inhibitor of porcine alpha-amylase (Ki = 11 +/- 2 nM) when a disaccharide is used as a substrate of alpha-amylase. However, the inhibition mode becomes complex when larger (> or = 7 saccharide units) alpha-amylase substrates are used. A second saccharide binding site on porcine alpha-amylase may enable larger oligosaccharides to displace RBI from its binding site in an intramolecular reaction.

The alpha-amylase from the yellow meal worm: complete primary structure, crystallization and preliminary X-ray analysis.

The alpha-amylase from Tenebrio molitor larvae (TMA) was purified from a crude larval extract. After removal of the N-terminal pyroglutamate residue and identification of the following 17 residues by Edman sequencing, the cDNA of mature TMA was cloned from larval mRNA. The encoded enzyme consists of 471 amino acid residues and has 57-79% sequence identity to other insect alpha-amylases and also shows high homology to the mammalian enzymes. TMA was crystallized in form of well-ordered orthorhombic crystals of space group P2(1)2(1)2(1) diffracting beyond 1.6 A resolution with unit cell dimensions of a = 51.24 A, b = 93.46 A, c = 96.95 A. TMA may serve as model system for the future analysis of interactions between insect alpha-amylase and proteinaceous plant inhibitors on the molecular level.

Endoproteinase-protein inhibitor interactions.

Nature uses protein inhibitors as important tools to regulate the proteolytic activity of their target proteinases. Most of these inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with their active-sites in a substrate-like "canonical" manner via an exposed reactive-site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors, two cysteine proteinase inhibitors, and three zinc endopeptidase inhibitors have been characterized in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are briefly discussed, with particular emphasis on the interaction between matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors of metalloproteinases (TIMPs).

Engineering of selective TIMPs.

Differences in proteinase susceptibility between free TIMP-1 and the TIMP-1-MMP-3 complex and mutagenesis studies suggested that the residues around the disulfide bond between Cys1 and Cys70 in TIMP-1 may interact with MMPs. The crystal structure of the complex between TIMP-1 and the catalytic domain of MMP-3 has revealed that the alpha-amino group of Cys1 bidentately chelates the catalytic zinc of MMP-3 and the Thr2 side chain occupies the S1' pocket. Generation of the N-terminal domain of TIMP-1 (N-TIMP-1) variants with 15 different amino acid substitutions for Thr2 has indicated that the nature of the side chain of residue 2 has a major effect on the affinity of N-TIMP-1 for three different MMPs (MMPs-1, -2 and -3). The results also demonstrate that the mode of binding of N-TIMP-1 residue 2 differs from the binding of the P1' residue of a peptide substrate.

Insights into MMP-TIMP interactions.

The proteolytic activity of the matrix metalloproteinases (MMPs) involved in extracellular matrix degradation must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance can result in serious diseases such as arthritis and tumor growth and metastasis. Knowledge of the tertiary structures of the proteins involved in such processes is crucial for understanding their functional properties and to interfere with associated dysfunctions. Within the last few years, several three-dimensional structures have been determined showing the domain organization, the polypeptide fold, and the main specificity determinants of the MMPs. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. Very recently, structural information also became available for some TIMP structures and MMP-TIMP complexes, and these new data elucidated important structural features that govern the enzyme-inhibitor interaction.

The interaction of metal ions with nucleic acids. NMR, EPR, CD and spectrophotometric study of the copper (II) interaction with 6-ketopurine ribosides.

The copper (II)-inosine system in water-DMSO solutions was investigated as a function of pH and the molar ratio between the ligand and copper(II) ion by the EPR, NMR, CD and visible absorption spectrometric methods. It was concluded that a simple M.[-N]L copper(II)-inosine 1:1 complex is formed over the pH range 1.4-5.0, while M.[-N]L2 complexes are present in the solutions of pH 5.0-6.2. From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex M2.(OH)2.[-N]L4 dominates. At pH values of 7.8-9.2 an insoluble, oxybridged species (M.O.[-N]L)n is formed in addition to the soluble paramagnetic M.[-N-1)L4 complex. Above pH 9.1 the nitrogenbridged polymeric complex (M.[-N-1].M[-N-7] )n is formed which is stable up to pH 12.5, and above pH 12.5 the only species found is the M.[-OH]L2 chelate complex in which inosine is coordinated to copper through the two ionized hydroxyl groups.

Interaction of metal ions with nucleic acids. Interaction of copper(II) with pyrimidine nucleosides and their derivatives.

1. In aqueous and non-aqueous solutions, copper(II) interacts with the N-3 of cytidine but not with the carbonyl group oxygens of pyrimidine nucleosides. 2. In aqueous solution, copper(II) interacts with the phosphate group and ribose of pyrimidine nucleotides, and additionally with N-3 of 5'-CMP. 3. Broadening of resonance signals of the H-5 proton of 5'-UMP and C-5 of 5'-UMP and 5'-TMP results probably from the interaction between metal ion and the phosphate group situated in direct vicinity of the above atoms. 4. In the copper(II)-pyrimidine nucleotide complexes in solid state, copper is coordinated with the phosphate group, and in 5'-CMP additionally with the pyrimidine moiety of the nucleotide.

Interaction of metal ions withe nucleic acids. Interaction of copper(II) with xanthosine and its derivatives.

It was found that in the interaction of xanthosine with copper(II) nitrogen N-7, and not oxygen atoms of carbonyl groups, is involved. In aqueous solution of XMP, copper(II) interacts not only with N-7 and the phosphate group but also with ribose. In a crystalline copper(II)-XMP complex [Cu(C10H11O9N4P).H2O], only the phosphate group and N-7 are involved.

Interaction of metal ions with nucleic acids. Interaction of copper(II) with adenosine and its derivatives.

The interaction of copper(II) with adenosine, 2'-deoxyadenosine, 1-methyladenosine, 7-deazaadenosine and AMP was studied by spectroscopic and magnetochemical methods. In non-aqueous medium, copper(II) interacts with adenosine and AMP at N-7 and N-1, and with 1-methyladenosine at N-7 and N-3. The copper ion is not bound to the NH2 group. In aqueous solution, copper(II) interacts both with N-7 and N-1 of adenosine, and in AMP additionally with the phosphate group. The interaction of copper(II) with the heterocyclic part, but not withthe phosphate group, is dependent on the extent of protonation of the molecular. A crystalline AMP-copper(II) complex [Cu(C10H12N5O7P).(H2O)2] was obtained; the phosphate group and probably N-7 are involved in the complex formation.

Interaction of metal ions with nucleic acids. Interaction of copper(II) with guanosine and its derivatives.

Interaction of copper(II) with guanosine, 2'-deoxyguanosine, 1-methylguanosine, 7-methylguanosine and GMP was studied withe use of spectroscopic and magneto-chemical methods. The main site of copper(II) binding in guanosine is nitrogen N-7; participation of N-1 is not excluded. The involvement of carbonyl oxygen in copper binding or copper chelation to N-7 and 0-6 is rather unlikely. A crystalline complex of copper(II) with GMP [Cu(C10H12O8N5P) .(H2O)3] was obtained, and it was demonstrated that copper(II) is bound with N-7 and the phosphate group.

Interaction of metal ions with nucleic acids. Interaction of copper(II) with inosine and its derivatives.

1. The interaction of copper(II) with inosine, 2'-deoxyinosine, 1-methylinosine, 7-deazainosine, 6-methoxypurine riboside and IMP was examined. 2. Copper binds with the purine base of the nucleosides, and in IMP also with the phosphate group. Non interaction with ribose hydroxyl groups was observed. 3. In non-aqueous medium, the main site of copper binding is N-7 of inosine and 1-methylinosine, and additionally N-1 in 6-methoxypurine riboside. 4. In aqueous medium, coordination of copper with N-1 and N-7 of inosine and IMP is pH-dependent. 5. Formation of either a five-membered copper chelate with N-7 and oxygen at C-6, or a four-membered chelate of the type C(6)-O-Cu-N(1), is rather unlikely. 6. The crystalline copper-IMP complex [Cu(C10H11O8N4P).(H2O)] contains presumably two copper atoms coordinated in different manner. The phosphate group and N-7, but not the carbonyl oxygen, participate in the complex formation.