Carlos Fernandez-Catalan

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP‐2 and membrane‐type‐1 MMP (MT1‐MMP) forms a cell surface located ‘receptor’ involved in pro‐MMP‐2 activation. We have solved the 2.75 Å crystal structure of the complex between the catalytic domain of human MT1‐MMP (cdMT1‐MMP) and bovine TIMP‐2. In comparison with our previously determined MMP‐3–TIMP‐1 complex, both proteins are considerably tilted to one another and show new features. CdMT1‐MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active‐site cleft that might be important for interaction with macromolecular substrates. The TIMP‐2 polypeptide chain, as in TIMP‐1, folds into a continuous wedge; the A‐B edge loop is much more elongated and tilted, however, wrapping around the S‐loop and the β‐sheet rim of the MT1‐MMP. In addition, both C‐terminal edge loops make more interactions with the target enzyme. The C‐terminal acidic tail of TIMP‐2 is disordered but might adopt a defined structure upon binding to pro‐MMP‐2; the Ser2 side‐chain of TIMP‐2 extends into the voluminous S1′ specificity pocket of cdMT1‐MMP, with its Oγ pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP‐1 for MT1‐MMP compared with TIMP‐2 might be explained by a reduced number of favourable interactions.

Structural properties of matrix metalloproteinases

Abstract. Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. 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. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.

Insights into MMP-TIMP interactions.

ABSTRACT: 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.

Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2.

Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel β-sheet and one short α-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.

Structural basis for possible calcium-induced activation mechanisms of calpains.

Abstract The calpains form a growing family of structurally related intracellular multidomainal cysteine proteinases, which exhibit a catalytic domain distantly related to papain. In contrast to papain, however, their activity in most cases depends on calcium. The calpains are believed to play important roles in cytoskeletal remodeling processes, cell differentiation, apoptosis and signal transduction, but have also been implicated in muscular dystrophy, ischemia, traumatic brain injury, neurodegenerative diseases, rheumatoid arthritis and cataract formation. The best characterized calpains are the ubiquitously expressed and mcalpains, consisting of a common 30 kDa small Ssubunit (domains V and VI) and slightly differing 80 kDa large Lsubunits (domains I to IV). We have recently determined the 2.3 å structure of recombinant fulllength human mcalpain in the absence of calcium, which reveals that the catalytic domain and the two calmodulinlike domains, previously believed to represent the unique calcium switch, are not positioned adjacent to each other, but are separated by the ?sandwich domain III, which distantly resembles C2 domains. Although the catalytic domain of apocalpain is strongly disrupted compared to papain (which explains its inactivity in the absence of calcium), the crystal structure reveals several sites where calcium could bind, thereby causing a subdomain fusion to form a papainlike catalytic center. All current evidence points to the cooperative interaction of several calcium binding sites. Sites identified include the three EFhand binding sites in each calmodulinlike domain, the negatively charged segments arranged around the activesite cleft (provided by both catalytic subdomains), as well as an exposed acidic loop of domain III, whose charge compensation could allow the adjacent barrellike subdomain IIb to move toward the helical subdomain IIa. The Glyrich Schain Nterminus and the calciumloaded acidic loop could target the conventional calpains to cellular/nuclear membranes, thereby explaining their strongly reduced calcium requirement in vivo and in vitro in the presence of acidic phospholipids.

Structure of human cyclin-dependent kinase inhibitor p19INK4d: comparison to known ankyrin-repeat-containing structures and implications for the dysfunction of tumor suppressor p16INK4a

BACKGROUND The four members of the INK4 gene family (p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d)) inhibit the closely related cyclin-dependent kinases CDK4 and CDK6 as part of the regulation of the G1-->S transition in the cell-division cycle. Loss of INK4 gene product function, particularly that of p16(INK4a), is found in 10-60% of human tumors, suggesting that broadly applicable anticancer therapies might be based on restoration of p16(INK4a) CDK inhibitory function. Although much less frequent, defects of p19(INK4d) have also been associated with human cancer (osteosarcomas). The protein structures of some INK4 family members, determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray techniques, have begun to clarify the functional role of p16(INK4a) and the dysfunction introduced by the mutations associated with human tumors. RESULTS The crystal structure of human p19(INK4d) has been determined at 1.8 A resolution using multiple isomorphous replacement methods. The fold of p19(INK4d) produces an oblong molecule comprising five approximately 32-residue ankyrin-like repeats. The architecture of the protein demonstrates the high structural similarity within the INK4 family. Comparisons to other ankyrin-repeat-containing proteins (GABPbeta, 53BP2 and myotrophin) show similar structures with comparable hydrogen-bonding patterns and hydrophobic interactions. Such comparisons highlight the splayed beta-loop geometry that is specific to INK4 inhibitors. This geometry is the result of a modified ankyrin structure in the second repeat. CONCLUSIONS Among the INK4 inhibitors, the highest amino acid sequence conservation is found in the helical stacks; this conservation creates a conserved beta-loop geometry specific to INK4 inhibitors. Therefore, in addition to models which predict that the conserved helix alpha6 is responsible for CDK inhibition, a binding mode whereby the loops of INK4 proteins bind to the CDKs should also be considered. A similar loop-based interaction is seen in the complex formed between the ankyrin-repeat-containing protein GABPbeta and_GABPalpha. This mode of binding would be consistent with the observation that p16(INK4a) is sensitive to deleterious mutations found throughout this tumor suppressor protein; these mutations probably destabilize the three-dimensional structure.

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).

Flexibility analysis and structure comparison of two crystal forms of calcium-free human m-calpain

Abstract The calpains form a growing family of structurally related intracellular multidomain cysteine proteinases containing a papainrelated catalytic domain, whose activity depends on calcium. The calpains are believed to play important roles in cytoskelatel remodeling processes, cell differentiation, apoptosis and signal transduction, but are also implicated in a number of diseases. Recent crystal structures of truncated rat and fulllength human apomcalpain revealed the domain arrangement and explained the inactivity of mcalpain in the absence of calcium by a disrupted catalytic domain. Proteolysis studies have indicated several susceptible sites, in particular in the catalytic subdomain IIb and in the following domain III, which are more accessible to attacking proteinases in the presence than in the absence of calcium. The current view is that mcalpain exhibits a number of calcium binding sites, which upon calcium binding cooperatively interact, triggering the reformation of a papainlike catalytic domain, accompanied by enhanced mobilisation of the whole structure. To further analyse the flexibility of mcalpain, we have determined and refined the human fulllength apomcalpain structure of a second crystal form to 3.15 å resolution. Here we present this new structure, compare it with our first structure now rerefined with tighter constrain parameters, discuss the flexibility in context with the proteolysis and calcium binding data available, and suggest implications for the calciuminduced activation process.

Crystallization and preliminary X-ray analysis of recombinant full-length human m-calpain.

m-Calpain constitutes the prototype of the superfamily of neutral calcium-activated cysteine proteinases. It is a heterodimer consisting of an 80 and a 30 kDa subunit. Recombinant full-length human m-calpain has been crystallized using macro-seeding techniques and vapour-diffusion methods. Two different monoclinic crystal forms (space group P2(1)) were obtained from a solution containing polyethylene glycol (M(W) = 10 000) as a precipitating agent. Complete data sets have been collected to 2.3 and 3.0 A resolution using cryo-cooling conditions and synchrotron radiation. The unit-cell parameters are a = 64.86, b = 133.97, c = 78.00 A, beta = 102.43 degrees and a = 51.80, b = 171.36, c = 64.66 A, beta = 94.78 degrees, respectively. The V(m) values indicate that there is one heterodimer in each asymmetric unit.