Veronika Harmat

MASP-1, a promiscuous complement protease: structure of its catalytic region reveals the basis of its broad specificity.

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 is an abundant component of the lectin pathway of complement. The related enzyme, MASP-2 is capable of activating the complement cascade alone. Though the concentration of MASP-1 far exceeds that of MASP-2, only a supporting role of MASP-1 has been identified regarding lectin pathway activation. Several non-complement substrates, like fibrinogen and factor XIII, have also been reported. MASP-1 belongs to the C1r/C1s/MASP family of modular serine proteases; however, its serine protease domain is evolutionary different. We have determined the crystal structure of the catalytic region of active MASP-1 and refined it to 2.55 Å resolution. Unusual features of the structure are an internal salt bridge (similar to one in factor D) between the S1 Asp189 and Arg224, and a very long 60-loop. The functional and evolutionary differences between MASP-1 and the other members of the C1r/C1s/MASP family are reflected in the crystal structure. Structural comparison of the protease domains revealed that the substrate binding groove of MASP-1 is wide and resembles that of trypsin rather than early complement proteases explaining its relaxed specificity. Also, MASP-1’s multifunctional behavior as both a complement and a coagulation enzyme is in accordance with our observation that antithrombin in the presence of heparin is a more potent inhibitor of MASP-1 than C1 inhibitor. Overall, MASP-1 behaves as a promiscuous protease. The structure shows that its substrate binding groove is accessible; however, its reactivity could be modulated by an unusually large 60-loop and an internal salt bridge involving the S1 Asp.

C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease.

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded β-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel “sandwich” mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.

Monospecific Inhibitors Show That Both Mannan-binding Lectin-associated Serine Protease-1 (MASP-1) and -2 Are Essential for Lectin Pathway Activation and Reveal Structural Plasticity of MASP-2

Background: MASP-1 is considered as auxiliary, whereas MASP-2 is considered as a key protease in lectin-pathway activation. Results: MASP-1 inhibitor SGMI-1 and MASP-2 inhibitor SGMI-2 completely block lectin pathway activation; the MASP-2·SGMI-2 complex reveals structural plasticity. Conclusion: MASP-1 is a key component; MASP-2 functions through induced fit or conformational selection. Significance: The lectin pathway activation model is incorrect. SGMIs revolutionize studying and enable regulating the lectin pathway. The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.

Flexible segments modulate co-folding of dUTPase and nucleocapsid proteins

The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting the two domains that are invisible in electron density maps. Small-angle X-ray scattering reveals that upon oligonucleotide binding the NC domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicate that fusion with NC domains dramatically alters the conformation of the flexible C-terminus by perturbing the orientation of a critical β-strand. Consequently, the C-terminal segment is capable of double backing upon the active site of its own monomer and stabilized by non-covalent interactions formed with the N-terminal segment. This co-folding of the dUTPase terminal segments, not observable in other homologous enzymes, is due to the presence of the fused NC domain. Structural and genomic advantages of fusing the NC domain to a shortened dUTPase in betaretroviruses and the possible physiological consequences are envisaged.

Quantitative characterization of the activation steps of mannan-binding lectin (MBL)-associated serine proteases (MASPs) points to the central role of MASP-1 in the initiation of the complement lectin pathway.

Background: Autoactivation of initiator proteases of complement is a two-step process. Results: Autoactivation and possible cross-activation steps of complement lectin pathway proteases were quantified. Conclusion: Only MASP-1 can autoactivate rapidly, and MASP-2 is activated by MASP-1. Significance: The determined kinetic data are helpful to interpret activation scenarios for the lectin pathway, and the presented strategy can be used to quantify autoactivation of other proteases. Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.

Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase

Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π–π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis.

Directed Evolution Reveals the Binding Motif Preference of the Lc8/Dynll Hub Protein and Predicts Large Numbers of Novel Binders in the Human Proteome

LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S]-4K-3X-2[T/V/I]-1Q0[T/V]1[D/E]2. The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V-5S-4R-3G-2T-1Q0T1E2 resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes.

Efficient synthesis and resolution of (±)-1-[2-carboxy-6-(trifluoromethyl)phenyl]pyrrole-2-carboxylic acid

Abstract A novel, efficient synthesis and resolution of (±)-1-[2-carboxy-6-(trifluoromethyl)-phenyl]pyrrole-2-carboxylic acid has been developed for the preparation of new members of optically active atropisomers. The e.e. values were determined by a highly sensitive 19F NMR spectroscopic method using β-cyclodextrin as chiral complexing agent. Single-crystal X-ray structures of the two diastereoisomeric salts and consequently, the absolute configurations of the enantiomers are also reported.

Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.

Structural Evidence for Non-Canonical Binding of Ca2+ to a Canonical EF-Hand of a Conventional Myosin.

We have previously identified a single inhibitory Ca2+-binding site in the first EF-hand of the essential light chain of Physarum conventional myosin (Farkas, L., Malnasi-Csizmadia, A., Nakamura, A., Kohama, K., and Nyitray, L. (2003) J. Biol. Chem. 278, 27399–27405). As a general rule, conformation of the EF-hand-containing domains in the calmodulin family is “closed” in the absence and “open” in the presence of bound cations; a notable exception is the unusual Ca2+-bound closed domain in the essential light chain of the Ca2+-activated scallop muscle myosin. Here we have reported the 1.8 Å resolution structure of the regulatory domain (RD) of Physarum myosin II in which Ca2+ is bound to a canonical EF-hand that is also in a closed state. The 12th position of the EF-hand loop, which normally provides a bidentate ligand for Ca2+ in the open state, is too far in the structure to participate in coordination of the ion. The structure includes a second Ca2+ that only mediates crystal contacts. To reveal the mechanism behind the regulatory effect of Ca2+, we compared conformational flexibilities of the liganded and unliganded RD. Our working hypothesis, i.e. the modulatory effect of Ca2+ on conformational flexibility of RD, is in line with the observed suppression of hydrogen-deuterium exchange rate in the Ca2+-bound form, as well as with results of molecular dynamics calculations. Based on this evidence, we concluded that Ca2+-induced change in structural dynamics of RD is a major factor in Ca2+-mediated regulation of Physarum myosin II activity.