A specificitás és gátlás szerkezeti alapjai az immunrendszer néhány fontos fehérjéjében = Structural basis of specificity and inhibition in some important proteins of the immune system

A komplementrendszer kezdeti proteázainak szerkezetét és tanulmányoztuk röntgen-krisztallográfával, specificitásuk és működésük során kialakuló kölcsönhatásaik atomi szintű jellemzése céljából. A fehérje kölcsönhatásokat a fehérjék egy szélesebb körén is jellemzzük kalmodulin, prolil-oligopeptidáz és acilpeptid-hidroláz szerkezetek segítségével. A MASP-1, MASP-2 és C1r komplement fehérjék esetén a komplexálatlan, komplexált és proenzim formák szerkezetének összehasonlítása volt a célunk. Meghatároztuk a MASP-1 aktív fragmentumának szerkezetét, amelyre jellemző széles szubsztrátkötő árok összefüggésben lehet a rokon komplement fehérjéknél szélesebb specificitásával. A C1r aktív katalitikus fragmentumának szerkezete alapján felülvizsgáltuk a C1r aktivációs mechanizmusát. A MASP-2 autoaktivációs komplexében és specifikus fehérje-inhibitorral alkotott komplexének szerkezetében a szerin proteázok kanonikus enzim/termék ill. enzim/szubsztrát kölcsönhatásain kívül kiterjedt kötőrégiók találhatók. A vizsgált enzimek szűk specificitása szempontjából fontos a kötőrégió kiterjedtsége, valamint a különböző komplexek képződésekor indukált konformáció változások-az enzim szerkezetének adaptálódása. | We studied structures of the early proteases of the complementusing crystallographic methods in order to characterize their specificity and interactions at atomic level. We used calmodulin, prolyl oligopeptidase and acylpeptide hydrolase strucutres for characterizing several aspects protein interactions. We studied the uncomplexed, complexed and proenzyme forms of MASP-1, MASP-2 and C1r. We solved the structure of the active fragment of MASP-1. The wide substrate binding groove characteristic to the structure is likely to be the structural basis of its specificity being wider than that of its related complement proteases. We revisited the autoactivation mechansim of C1r autoactivation, based on the structure of its active catalytic fragment. The structures of the autoactivation complex of MASP-2 and its complex with a specific serine-protease inhibitor revealed extended contact regions beyond the canonical enzyme/product and enzyme/inhibitor interactions. The extensions of the binding region and conformational adaptation of these enzymes are likely to be major bases of their narrow specificity

Theoretical Aspects of Molecular Recognition

Molecular recognition is a key process in non-covalent interactions, which determines, among others, host-guest complexation, drug action and protein-protein interaction. A simple and attractive formulation is the lock-and-key analogy defining the host as a lock accommodating the guest as a key. We stress three major aspects of molecular recognition, determining both complementarity between host and guest and similarity within a group of guest molecules. These aspects are: steric, i.e. maximization of close contacts, electrostatic, i.e. maximization of electrostatic attraction between host and guest, as well as hydrophobic, i.e. avoiding hydrophobic hydration, which can be reached by the maximization of apolar contacts between interacting molecules. Some examples are presented from our laboratory: the complexes of acylaminoacyl peptidase with small peptides, the effect of heparin binding on inhibitory potency of C1- inhibitor as well as small-molecule ligand binding to prolyl oligopeptidase and calmodulin

Structural Diversity in Calmodulin - Peptide Interactions

Calmodulin (CaM) is a highly conserved eukaryotic Ca2+ sensor protein that is able to bind a large variety of target sequences without a defined consensus sequence. The recognition of this diverse target set allows CaM to take part in the regulation of several vital cell functions. To fully understand the structural basis of the regulation functions of CaM, the investigation of complexes of CaM and its targets is essential. In this minireview we give an outline of the different types of CaM - peptide complexes with 3D structure determined, also providing an overview of recently determined structures. We discuss factors defining the orientations of peptides within the complexes, as well as roles of anchoring residues. The emphasis is on complexes where multiple binding modes were found

Crystallization and preliminary crystallographic analysis of dUTPase from the helper phage Φ11 of Staphylococcus aureus

Staphylococcus aureus superantigen-carrying pathogenicity islands (SaPIs) have a determinant role in spreading virulence genes among bacterial populations that constitute a major health hazard. Repressor (Stl) proteins are responsible for transcriptional regulation of pathogenicity island genes. Recently, a derepressing interaction between the repressor Stl SaPIbov1 with dUTPase from the Φ11 helper phage was suggested [Tormo-Mas et al. (2010). Nature 465, 779-782]. Towards elucidating the molecular mechanism of this interaction, this study reports expression, purification, and X-ray analysis of Φ11 dUTPase that contains a phage-specific polypeptide segment not present in other dUTPases. Crystals were obtained using the hanging-drop vapor-diffusion method at room temperature. Data were collected from one type of crystal to 2.98 Å resolution. The crystal of Φ11 dUTPase belonged to the cubic space group I23, with unit-cell parameters a=98.16 Å, α=β=γ= 90.00o

Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions.

Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well

A fehérjefeltekeredés alapjai: a feltekeredés, a letekeredés és az aggregáció vizsgálata atomi szintű kísérleti és számítási módszerekkel = Unifying principles of protein folding: understanding folding, unfolding and aggregation at atomic-level by experimental and computational methods

Célkitűzésünk a fehérjefeltekeredés és konformációs állapotváltozások mélyebb megértése, alkalmasan megválasztott modellrendszerek - azaz különböző méretű és belső dinamikájú fehérjék - vizsgálatán keresztül. A kémiailag vagy bakteriális úton előállított fehérjék szerkezeti és dinamikai paramétereinek meghatározását követően olyan új variánsokat tervezünk, amelyek vagy még rendezettebbek, vagy a belsőleg rendezetlen fehérjék esetében, még rendezetlenebbé váltak. A vizsgált mini-, moduláris fehérjék, valamint a funkcionálisan rendezetlen rendszerek körét olyan fontos molekulák alkották, amelyek közvetlen kapcsolatban állnak a cukorbetegség, az agyi neurodegeneratív elváltozások, vagy az immunológia különböző tárgykörével. Kutatásaink során mind a téralkat, mind a fehérjék belső mozgékonyságának explicit jellemzésén keresztül értettük meg jobban az adott makromolekulák biológiai szerepét, fiziológiás jelentőségét. A peptidek és fehérjék racionális tervezése során nem csak mutánsokat és variánsokat, de nem-természetes aminosavakból felépülő „foldamereket” is terveztünk, majd állítottunk elő. Vizsgáltuk a béta-aminosavak és béta-peptidek beépíthetőségét és felhasználhatóságát a téralkat és mozgékonyság függvényében. Eredményeinket 33 angol nyelvű közleményben (IF=116,984), webszervereken, és több mint 40 hazai és nemzetközi tudományos fórumon és konferencián adtuk közre. | Our aim was to decipher and better understand the molecular details of protein folding and conformational switching achieved via selected polyamide nanosystems of different size and internal dynamics. Once the structural parameters of the chemically synthesized or bacterially expressed proteins were determined, new variants and mutants were designed. Thus, the polypeptide chain was made either more ordered for globular proteins, or less structured for the disordered proteins (IDP). The investigated mini-, modular, globular and unstructured proteins are involved in biologically important cellular processes associated with either Diabetes Mellitus, neurodegenerative or immunological diseases. Our goal was to better understand the biological role and function of these proteins by monitoring both their structure (NMR and X-ray crystallography) and internal dynamics (NMR). The rational design of selected peptides and proteins resulted in not only mutants and variants of the parent macromolecule, but also foldamers. The applicability of beta amino acids and beta peptides were in focus. Our results were published in international journals (33 articles, IF=116,984), webservers, and presented on over 40 scientific meetings

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

Achieving Functionality Through Modular Build-up: Structure and Size Selection of Serine Oligopeptidases

Enzymes of the prolyl oligopeptidase family (S9 family) recognize their substrates not only by the specificity motif to be cleaved but also by size - they hydrolyze oligopeptides smaller than 30 amino acids. They belong to the serine-protease family, but differ from classical serine-proteases in size (80 kDa), structure (two domains) and regulation system (size selection of substrates). This group of enzymes is an important target for drug design as they are linked to amnesia, schizophrenia, type 2 diabetes, trypanosomiasis, periodontitis and cell growth. By comparing the structure of various members of the family we show that the most important features contributing to selectivity and efficiency are: (i) whether the interactions weaving the two domains together play a role in stabilizing the catalytic triad and thus their absence may provide for its deactivation: these oligopeptidases can screen their substrates by opening up, and (ii) whether the interaction-prone β-edge of the hydrolase domain is accessible and thus can guide a multimerization process that creates shielded entrance or intricate inner channels for the size-based selection of substrates. These cornerstones can be used to estimate the multimeric state and selection strategy of yet undetermined structures

Miozin motorfehérjék: szerkezet, funkció, szabályozás, kötőpartnerek = Myosin motor proteins: structure, function, regulation, binding partners

A pályázat során a miozin motorfehérjék szerkezet-funkció összefüggéseit vizsgáltuk. A pályázat talán legfontosabb eredménye, hogy elsőnek sikerült atomi felbontású képet kapnunk a miozin coiled-coil (alfa-helikális dimer) szerkezetű farok régió egy részletéről, ráadásul egy olyan instabil doménről, amely a konvencionális miozin (II-es típus) működésében és szabályozásában is fontos szerepet játszhat. A szabályozás kapcsán továbbá meghatároztuk egy Ca2+-ionokkal gátolt miozin II regulációs doménjánek térszerkezetét. Egy, a miozin motor doménben található, régóta ismert reaktív lizin oldalláncról bizonyítottuk, hogy konformációs szenzorként alkalmazható az összes miozin II-ben. Érdekes új eredménynek tekintjük, hogy a VI-os típusú miozin farok régiójában azonosítottunk egy olyan, erősen töltött molekula-részletet, amely egyszálú stabil alfa-hélixet alkot. Ezt a szerkezeti motívumot in silico módszerekkel sok más fehérjében is megtaláltuk. Az V-ös típusú, intracelluláris transzporter-ként működő miozin Drosophila ortológjának ATPáz kinatikáját vizsgálva azt találtuk, hogy a gerinces miozin V-től eltérően ez a motorfehérje nem processzív működésű. A gerinces miozin V-nél a teher kötésében résztvevő adapter molekula, az ún. dinein könnyű lánc kötőhelyét azonosítottuk, és a dimer farok régióra gyakorolt stablizáló hatását mutattuk ki. | In this project we have conducted structure-function studies on myosin motor proteins. Perhaps the most important result is that we have obtained the first atomic resolution picture of the coiled-coil (alpha-helical dimer) myosin tail, and what is more the structure of an unstable domain having important role in the function and regulation of conventional myosin (type II). Regarding regulation, we have determined the 3-dimensional structure of a regulatory domain from a Ca2+-inhibited myosin II. We have proved that a long-time known reactive lysine of the motor domain can be used as a conformational sensor in all of myosin IIs. An interesting new result is that we have identified a highly charged segment in the tail domain of myosin VI that forms a stable single stranded alpha-helix. This structural motif has been found in several other proteins by in silico methods. Studying the ATPase kinetics of Drosophila myosin V, we have shown that this motor is not processive unlike its vertebrate orthologs. We have localized the binding site of the dynein light chain, a cargo binding adapter subunit, on vertebrtae myosin V, and have shown that it stabilizes the structure of the dimeric tail domain