MASP-2

MASP-2 is responsible for the first enzymatic event of the lectin pathway of the complement cascade - a major element of innate immunity. MASP-2 is a modular enzyme with restricted specificity. It is capable of autoactivation, moreover the proenzyme form shows catalytic activity on natural substrate. We solved the structure of the zymogen and activated forms of the enzyme. Comparison of the active structure with C1s the analogous enzyme of the classical pathway of complement reveals that nearly identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions. We also focused on the structural background of the ability of the zymogen form of MASP-2 to undergo autoactivation. The structure of the zymogen form of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes upon activation. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form.

Model of the enzyme-substrate complex of zymogen and active MASP-2.

Cooperation: Dr. Péter Závodszky and Dr. Péter Gál,

Calmodulin-ligand complexes

Calmodulin (CaM), a key player in intracellular Ca2+-signal transduction. It is a small protein containing two Ca2+-binding EF-hand motives in its both domains. We studied its complexes with small molecular ligands taking different effects on calmodulin action. An extraordinarily high affinity arylalkylamine-type calmodulin antagonist competes for the hydrophobic pockets of CaM with target proteins or with the classical CaM antagonist, trifluoperazine (TFP). Its complex with CaM shows extended contacts burying large hydrophobic surfaces. We solved the crystal structure of CaM complexed with a derivative of the anticancer drug vinblastine (KAR-2) and studied the complex in solution by NMR spectroscopy. In contrast to vinblastine KAR-2 does not have antagonistic effect on CaM-modulated processes, though they show similar CaM affinity. KAR-2 binds in a region of CaM overlapping only partially with the binding sites of the target proteins. That explains why KAR-2 does not prevent calmodulin from binding most of its physiological targets.

KAR-2 binding by calmodulin

Cooperation: Dr. Judit Ovádi, Ovádi group, Institute of Enzymology Budapest
Dr. Zsolt Böcskei, Chinoin Pharmaceuticals, Budapest

Publications
István Horváth, Veronika Harmat, András Perczel, Villõ Pálfi, László Nyitray, Attila Nagy, Emma Hlavanda, Gábor Náray-Szabó and Judit Ovádi:
The structure of calmodulin's complex with KAR-2: A novel mode of binding explains the drug's unique pharmacology
J. Biol. Chem. (2005) 280(9):8266-8274.
[abstract]

Harmat V, Böcskei Z, Náray-Szabó G, Bata I, Csutor AS, Hermecz I, Arányi P, Szabõ B, Liliom K, Vértessy BG, Ovádi J A new potent calmodulin antagonist with arylalkylamine structure: Crystallographic, spectroscopic and functional studies J. Mol. Biol. 297: 747-755 (2000)
[abstract]

Structures determined
CaM - 2 TFP complex: [1A29], CaM - AAA complexes: [1QIV] and [1QiW], CaM - KAR-2 complex: [1XA5]

Complex of crayfish trypsin and SGTI

We solved the crystal structure of crayfish trypsin complexed with SGTI, a taxon selective trypsin inhibitor from desert locust Schistocerca gregaria. The SGTI binding region is extended contacting the P12-P5' residues of the inhibitor. Comparison of the structure of SGTI in the complex and its solution structure (our previous NMR studies) reveals two regions undergoing major conformation change allowing shape adaptation of the inhibitor in the complex. The structure represents the Michaelis complex to atomic resolution (1.2 Angstrom) and ensures deeper insight in serine protease catalysis. Comparison with the acyl-enzyme intermediate of a related enzyme (Katona et al. JBC 277, 21962, 2002) reveals that the strength of the enzyme-substrate hydrogen bonds changes during catalysis.

Crayfish trypsin - SGTI complex

Cooperation: Prof. László Gráf, Department of Biochemistry, Eötvös Loránd University, Budapest,
Dr. Gergely Katona, Department of Biochemistry, University of Leicester, UK

Publications
Fodor K, Harmat V, Neutze R, Szilagyi L, Graf L, Katona G:
Substrate hydrogen bond shortening during the acylation phase of serine protease catalysis Biochemistry 45: 2114-2121 (2006)
[abbstract]

Krisztián Fodor, Veronika Harmat, Csaba Hetényi, József Kardos, József Antal, András Perczel, András Patthy, Gergely Katona and László Gráf:
Extended intermolecular interactions in a serine protease canonical inhibitor complex account for strong and highly specific inhibition
J. Mol. Bio. (2005) 350:156-169.
[abstract]

Structure determined
Crayfish trypsin - SGTI complex: [2F91]

Myosin regulation domain

Class II myosins have a fundamental role in muscle contraction and in a variety of cellular motilities. The regulatory domain (RD) acts as a lever arm during force generation, and it is the site of regulation in regulated conventional myosins. The class II myosin of Physarum polycephalum is uniquely inhibited by direct binding of Ca2+. In our structure of the regulatory domain, the regulatory Ca2+ is bound by the ELC. The Ca2+-binding first EF-hand unusually possesses "closed" conformation. In scallop myosin the activating Ca2+ is bound to the first EF-hand of the ELC that is also in a closed state, but it is coordinated by different residues. In the case of Physarum myosin II the change in dynamics of the molecule upon Ca2+-binding plays an important role in Ca2+-inhibition.

Structure of the myosin regulatory domain

Cooperation: Dr. László Nyitray,

Structure determination of N-trimethylsilylated cyclic ureas

We solved the crystal structures of N-trimethylsilylated cyclic ureas with 5- and 6-membered rings. The bond lengths and angles around the silicon atom vary significantly, supporting that the silylating power depends on the pseudo-pentacoordinate character of the tetracoordinate silicon. A simple structure-based number measuring the pseudo-pentacoordinate character of the silicon can be used for predicting reactivity.

Cooperation: Dr. Roland Szalay and Dr. Dezsõ Kanusz, Department of General and Inorganic Chemistry; Dr. Gábor Pongor, Department of Theoretical Chemistry, Eötvös Loránd University, Budapest; Dr. Zsolt Böcskei,