Konformációs viszonyok és elektronszerkezet-változások vizsgálata fehérjékben = Investigation of conformational fluctuations and electronic structure variations in proteins

Feltérképeztük a foszfoglicerát kináz molekuláris felismerési folyamataiban szerepet játszó konformációs változásokat. Elvégeztük a foszfoinozitol kináz 3 alfa izoformájánnak 3D szerkezetpredikcióját, azonban a ligandumkötődés vizsgálatára a kooperáló partner elállása miatt nem került sor. Felderítettük a tetrahidribiopterin (BH4) kofaktor szerepe a nitrogén monoxid szintetáz (NOS) aktiválódásában. Meghatároztuk a NO kötőhelyét a nitroforin 4 (NP4) fehérje aktív helyén. Low-mode (LMOD) keresésen alapuló platfromfüggetlen konformerkereső és dokkoló eljárást dolgoztunk ki, amely az AMBER programcsomagban is hozzáférhető. Elvégeztük a kifejlesztett eljárás több konformerkereső módszerrel történő összehasonlító vizsgálatát. Eljárást dolgoztunk ki humán P450 2C9 ligandumok azonosítására és az izoforma specificitás vizsgálatára a 2C családban. Felderítettük a NADH kofaktor és NO kölcsönhatás szerepét a P450 NO-reduktáz fehérjében. A támogatott időszak alatt megkezdett hisztamin receptorok kutatását érintő vizsgálataink alapján új lipofil zsebet találtunk a H1 receptorban, ligandum információkkal segített homológiamodellezéssel előállítottuk a H4 receptor első atomi felbontású modelljét és eljárást dolgoztunk ki új H4 ligandunok azonosítására. Utóbbi eljárást az eddig ismert legkiterjedtebb szerkezet alapú szűrővizsgálatban alkalmazva új, kísérletileg is megerősített H4 ligandumokat azonosítottunk. | Conformational motions responsible for the substrate recognition in phosphoglycerate kinase have been explored. Homology model for phosphoinositol 3 kinase alpha isoform has been developed, however docking studies were suspended due to the changed interest of the partner (ComGenex Inc). The role of tetrahydrobiopterin cofactor in nitric oxide synthase has been clarified. The putative nitric oxide binding site in nitrophorin 4 has been identified. A platform-independent LMOD conformational search method has been developed and integrated to AMBER package at UCSF. The performance of this method has been evaluated and compared to other algorithms. A new virtual screening protocol has been developed for the identification of CYP 2C9 ligands. The protocol was useful for isofom specificity studies in the 2C family. The role of NADH-nitric oxide interaction in P450-No reductase has been investigated. A new lipophilic binding pocket in human histamine H1 receptor has been identified. The first atom-level model of human histamine H4 receptor has been constructed by ligand supported homology modelling. that was used to develop an effective virtual screening protocol. The protocol allowed us to perform the largest structure based virtual screening experiment using a screening database of more than 8 million compounds. Identified new chemical scaffolds showed submicromolar binding affinity towards the human histamine H4 receptor as revealed by experimental studies

Catalytically distinct states captured in a crystal lattice: the substrate-bound and scavenger states of acylaminoacyl peptidase and their implications for functionality

Acylaminoacyl peptidase (AAP) is an oligopeptidase that only cleaves short peptides or protein segments. In the case of AAP fromAeropyrum pernix(ApAAP), previous studies have led to a model in which the clamshell-like opening and closing of the enzyme provides the means of substrate-size selection. The closed form of the enzyme is catalytically active, while opening deactivates the catalytic triad. The crystallographic results presented here show that the open form of ApAAP is indeed functionally disabled. The obtained crystal structures also reveal that the closed form is penetrable to small ligands: inhibitor added to the pre-formed crystal was able to reach the active site of the rigidified protein, which is only possible through the narrow channel of the propeller domain. Molecular-dynamics simulations investigating the structure of the complexes formed with longer peptide substrates showed that their binding within the large crevice of the closed form of ApAAP leaves the enzyme structure unperturbed; however, their accessing the binding site seems more probable when assisted by opening of the enzyme. Thus, the open form of ApAAP corresponds to a scavenger of possible substrates, the actual cleavage of which only takes place if the enzyme is able to re-close.</jats:p

Fémek szerepe a fehérjeszerkezetben és - működésben = The role of metals in protein structure and function

Fehérjekrisztallográfia, mágneses magrezonancia-spektroszkópia és molekulamodellezés segítségével vizsgáltuk az összefüggéseket néhány metalloprotein, valamint egy új típusú, rendezetlen fehérje szerkezete és működése között. Hatékony módszert fejlesztettünk ki a reakcióút kvantummechanikai számítására enzimekben. Tisztáztuk a DNS javításában fontos szerepet játszó dUTPáz által katalizált reakció legtöbb részletét. Meggyőző bizonyítékokat szolgáltattunk arra, hogy az enzimatikus foszfáthidrolízis során a dUTPázban nagy energiájú, trigonális bipiramisos elrendeződésű intermedier keletkezik. Kimutattuk, hogy a KAR-2 nevű molekula más, biszindol típusú ligandumoktól eltérő módon kötődik a kalmodulinhoz, ez magyarázza különleges fiziológiai hatását. Elvégeztük a hemoglobin hem-csoportjainak normál koordináták szerinti analízisét, amiből következtetéseket vontunk le a szerkezetre vonatkozóan. A deformációk azt mutatják, hogy a hem csoport szerkezete érzékeny a molekula távoli részében kötődő effektor jelenlétére, ami az allosztérikus szabályozás hatásmechanizmusának a tercier szerkezettel való kapcsolatát támasztja alá. A közelmúltban egy új agy-specifikus fehérjét izoláltunk, melynek átlagos rendezetlensége 46-47%, tehát szerkezet nélkülinek tekinthető. Részletes vizsgálatokat végeztünk e fehérje, illetve különböző fehérjékkel képezett komplexe szerkezetére vonatkozóan. | We investigated the relationship between the structure and activity of some metalloproteins and a new unfolded protein. We developed an efficient method for the quantum mechanical calculation of the reaction path in enzymes. Most details of the reaction catalysed by dUTPase, playing an important role in DNA repair, have been clarified. We provided convincing evidence that during enzymatic phosphate hydrolysis a high-energy, trigonal bipyramidal intermediate is formed. We have shown that the molecule KAR-2, in contrast to other bisindole-type ligands, has a different binding mode to calmodulin, which explains its special physiological effect. We performed the normal co-ordinate analysis of the hem groups of haemoglobin and derived conclusions on their structure. The deformations indicate that the structure of the hem group is sensitive to the presence of an effector bound in a distant region of the molecule. This finding supports the relation between the allosteric mechanism of action and the tertiary structure. Recently we isolated a new brain-specific protein, which is 46 to 47 per cent disordered, i.e. it can be considered as unfolded. We made detailed studies on the structure of this protein and its complex with others

Szerkezeti biológia = Structural biology

Kiroptikai spektroszkópia (ECD, VCD) - Az abszolút konfiguráció meghatározása - Gyűrűs és lineáris peptid modellek konformációjának meghatározása - Királis diródium komplexek ECD és VCD vizsgálata Fehérje NMR és modellezés - Peptidek számitástechnikai vizsgálata - Fehérjék NMR vizsgálata Fehérje röntgenkrisztallográfia - Egy prolil-oligopeptidáz (POP) komplexei kristályszerkezetének meghatározása - A kalmodulin (CaM) vizsgálata - A molekuláris felismerés szerkezeti vonatkozásai - A Pyrococcus horikoshii acilamino-peptidáz (AAP) kristályszerkezetének meghatározása Biokémiai vizsgálatok - A scallop-peptid vizsgálata három kristályközegben - A miozin-6 utolsó (tail) doménje szerkezetének predikciója - A coiled-coil és rendezetlen fehérje szegmensek kereszt-predikciójának analízise - Öt természetes és két szintetikus, töltéssel rendelkező ?-hélix lánc mikroszekundum időskálájú molekuladinamikai szimulációja vizes közegben - Az LC8 dynein centrális (hub) fehérje könnyű láncának (DYNLL) vizsgálata Immunológiai vizsgálatok - Az SHP-2 tirozin-foszfatáz vizsgálata. Különböző szerkezetű foszfopeptidek tanulmányozása - Egy biotinilezett kollagén epitóp peptid, az extravidin-peptid complex és extravidin szerkezetvizsgálata Közlemény: 36 | Chiroptical spectroscopy (ECD, VCD) - Determination of the absolute configuration - Determination of the conformation of cyclic and linear model peptides - Studies on the chiral dirhodium complexes by ECD and VCD Protein NMR and protein modeling - Computational work on peptides. - NMR studies in proteins Protein X-ray crystallography - The crystal structures of prolyl oligopeptidase (POP) in complexes were solved. - Studies on calmodulin (CaM). - Structural aspects of molecular recognition were characterized. - The crystal structure of Pyrococcus horikoshii acylaminoacyl peptidase was solved. Biochemical studies - The structure of a scallop peptide in three crystal environments was determined . - The structure of the tail domain of myosin-6 was predicted. - A thorough analysis of cross-predictions of coiled-coil and disordered protein segments was performed. - Microsecond classical molecular dynamics simulations of five naturally occurring and two synthetic charged single ?-helices were performed. - Studies on LC8 dynein light chain (DYNLL), a hub protein. Immunological studies - Studies on SHP-2 tyrosine phosphatase. A variety of phosphopeptides were tested. - Structural studies were performed on biotinylated collagen epitope peptide, an extravidin-peptide complex and extravidin. Papers: 3

NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg²⁺ Ion

In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg2+-free and Mg²⁺-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg²⁺-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg²⁺-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an “NMR ensemble” based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg²⁺-bound and Mg²⁺-free states of G12C KRAS/GDP.ISSN:1422-006

The mutation‐dependent pathogenicity of NPHS2 p.R229Q

NPHS2, encoding podocin, is the major gene implicated in steroid‐resistant nephrotic syndrome. Its c.686G>A, p.R229Q variant is the first human variant with a mutation‐dependent pathogenicity; it is only pathogenic when trans‐associated to specific mutations. Secondary to its high allele frequency in the European, South Asian, African, and Latino populations, its benign trans‐associations can be accidentally identified in affected patients. Distinguishing pathogenic and benign p.R229Q associations can be challenging. In this paper, we present the currently known pathogenic and benign associations, and show that a rare p.R229Q association can be considered pathogenic if the variant in trans meets the following criteria; it affects the 270–351 residues and alters but does not disrupt the oligomerization, its p.R229Q association is found in a family with slowly progressing focal segmental glomerulosclerosis, but is expected to be rare in the general population (15% of the p.R229Q associations identified so far in patients are benign

C-terminal oligomerization of podocin mediates interallelic interactions

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit dia- phragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3′ mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283–382): principally through the first C-terminal helical region (H1, 283–313), which forms a coiled coil as shown by circular di- chroism spectroscopy, and through the 332–348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly in- fluence each other's localization (r2 = 0.68, P = 9.2 × 10 −32). The dominant negative effect resulting in in- tracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligo- merization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals