Identification of nitroglycerin‐induced cysteine modifications of pro‐matrix metalloproteinase‐9

Nitroglycerin (NTG), an important cardiovascular agent, has been shown recently to activate matrix metalloproteinase‐9 (MMP‐9) in biological systems, possibly leading to destabilization of atherosclerotic plaques. The chemical mechanism for this activation, particularly on the cysteine switch of the pro‐form of MMP‐9 (proMMP‐9), has not been investigated and was examined here using nano‐flow liquid chromatography coupled to mass spectrometry. In order to obtain high sequence coverage, two orthogonal enzymes (trypsin and GluC) were employed to digest the protein in parallel. Two complementary activation methods, collision‐induced dissociation (CID) and electron‐transfer dissociation (ETD), were employed for the identification of various modifications. A high‐resolution Orbitrap analyzer was used to enable confident identification. Incubation of NTG with proMMP‐9 resulted in the formation of an unstable thionitrate intermediate and oxidation of the cysteine switch to sulfinic and irreversible sulfonic acid derivatives. The unstable thionitrate modification was confirmed by both CID and ETD in the proteolytic peptides produced by both trypsin and GluC. Incubation of proMMP‐9 with diethylenetriamine NONOate (a nitric oxide donor) led to sulfonic acid formation, but no observable sulfinic acid modification. Extensive tyrosine nitration by NTG was observed at Tyr‐262, in close proximity to an oxidized Cys‐256 of proMMP‐9. The intramolecular interaction between these two residues toward NTG‐induced oxidation was examined using a synthesized peptide representing the sequence in this domain, PWCSTTANYDTDDR, and the modification status was compared against an analog in which Cys was substituted by Ala. We observed a thionitrate product, extensive Cys oxidative modifications and enhanced tyrosine nitration with the Cys peptide but not with the Ala analog. Our results indicated that neighboring Cys and Tyr residues can facilitate each other's oxidation in the presence of NTG. Copyright © 2011 John Wiley & Sons, Ltd.     

Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase

We report the 2.6 A X-ray crystal structure of a 190 kDa homodimeric fragment from module 3 of the 6-deoxyerthronolide B synthase covalently bound to the inhibitor cerulenin. The structure shows two well-organized interdomain linker regions in addition to the full-length ketosynthase (KS) and acyltransferase (AT) domains. Analysis of the substrate-binding site of the KS domain suggests that a loop region at the homodimer interface influences KS substrate specificity. We also describe a model for the interaction of the catalytic domains with the acyl carrier protein (ACP) domain. The ACP is proposed to dock within a deep cleft between the KS and AT domains, with interactions that span both the KS homodimer and AT domain. In conjunction with other recent data, our results provide atomic resolution pictures of several catalytically relevant protein interactions in this remarkable family of modular megasynthases.     

A proteome-wide analysis of domain architectures of prokaryotic single-spanning transmembrane proteins

We performed a proteome-wide survey of the domain architectures in single-spanning transmembrane (TM) proteins (single-spannings) from 87 sequenced prokaryotic (Bacterial and Archaean) genomes by assigning Pfam domains to their N-tail and C-tail loops. Out of 14,625 single-spannings, 3,516 sequences have at least one domain assigned, and no domains were assigned to 7,850, with the remaining 3,259 with less reliable assignment. In the domain-assigned sequences, 3116 sequences are with at most two domains, and the other 400 sequences with more than two. The assigned domains distribute over 651 Pfam families, which account for 11.4% of the total Pfam-A families. Among the 651 families are mostly soluble-protein-originated ones, but only 21 families are unique to TM proteins. The occurrence frequency of the individual domain families follows a power-law, that is, 264 families occur only once, 106 just twice, and the families appeared more than 30 times are counted by only 39. It is found that the great majority of the sequences having one or two domains are of the type II topology with the C-tail loop containing domains on it. On the contrary, the N-tail loop of the same type topology seldom carries domains. Importantly, the assigned domains are always found on the tail loops longer than 60 residues, even for the small domains with less than 30 residues. There are still as many as 5,800 sequences without assigned domains in spite of having at least one long tail, on which no less than 1,000 novel domain families are expected most likely to lie concealed unknown yet. We also investigated the domain arrangement preference and the domain family combination patterns in 'singlets' (single-spannings with one assigned domain) and 'doublets' (with two domains).     

RNA cleavage by a DNA enzyme with extended chemical functionality.

In vitro selection techniques were applied to the development of a DNA enzyme that contains three catalytically essential imidazole groups and catalyzes the cleavage of RNA substrates. Nucleic acid libraries for selection were constructed by polymerase-catalyzed incorporation of C5-imidazole-functionalized deoxyuridine in place of thymidine. Chemical synthesis was used to define a minimized catalytic domain composed of only 12 residues. The catalytic domain forms a compact hairpin structure that displays the three imidazole-containing residues. The enzyme can be made to cleave RNAs of almost any sequence by simple alteration of the two substrate-recognition domains that surround the catalytic domain. The enzyme operates with multiple turnover in the presence of micromolar concentrations of Zn2+, exhibiting saturation kinetics and a catalytic rate of >1 min-1. The imidazole-containing DNA enzyme, one of the smallest known nucleic acid enzymes, combines the substrate-recognition properties of nucleic acid enzymes and the chemical functionality of protein enzymes in a molecule that is small, yet versatile and catalytically efficient.     

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

Background  

We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity.     

Long-range interaction between heterogeneously charged membranes

Despite their neutrality, surfaces or membranes with equal amounts of positive and negative charge can exhibit long-range electrostatic interactions if the surface charge is heterogeneous; this can happen when the surface charges form finite-size domain structures. These domains can be formed in lipid membranes where the balance of the different ranges of strong but short-ranged hydrophobic interactions and longer-ranged electrostatic repulsion result in a finite, stable domain size. If the domain size is large enough, oppositely charged domains in two opposing surfaces or membranes can be strongly correlated by the electrostatic interactions; these correlations give rise to an attractive interaction of the two membranes or surfaces over separations on the order of the domain size. We use numerical simulations to demonstrate the existence of strong attractions at separations of tens of nanometers. Large line tensions result in larger domains but also increase the charge density within the domain. This promotes correlations and, as a result, increases the intermembrane attraction. On the other hand, increasing the salt concentration increases both the domain size and degree of domain anticorrelation, but the interactions are ultimately reduced due to increased screening. The result is a decrease in the net attraction as salt concentration is increased.     

Dynamics of water molecules in the active-site cavity of human cytochromes P450

We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot. In the simulations, the cavities are completely filled with water molecules, although with approximately 20% lower density than in bulk water. The 2A6 protein differs from the other three in that it has a very small cavity with only two water molecules and no exchange with the surroundings. The other three proteins have quite big cavities, with 41 water molecules on average in 2C8 and 54-58 in 2C9 and 3A4, giving a water volume of 1500-2100 A3. The two crystal structures of 2C9 differ quite appreciably, whereas those of 3A4 are quite similar. The active-site cavity is connected to the surroundings by three to six channels, through which there is a quite frequent exchange of water molecules (one molecule is exchanged every 30-200 ps), except in 2A6. Most of the channels are observed also in the crystal structures, but two to three channels in each protein open only during the simulations. There are no water molecules close to the heme iron ion in these simulations of the high-spin ferric state (the average distance to the closest water molecule is 3.3-5 A), and there are few ordered water molecules in the active sites, none of which is conserved in all proteins.     

Metallobiological necklaces: mass spectrometric and molecular modeling study of metallation in concatenated domains of metallothionein

The ubiquitous protein metallothionein (MT) has proven to be a major player not only in the homeostasis of Cu(I) and Zn(II), but also binds all the Group 11 and 12 metals. Metallothioneins are characterised by the presence of numerous cys-x-cys and cys-cys motifs in the sequence and are found naturally with either one domain or two, linked, metal-binding domains. The use of chains of these metal-thiolate domains offers the possibility of creating chemically tuneable and, therefore, chemically dependent electrochemical or photochemical surface modifiers or as nanomachinery with nanomechanical properties. In this work, the metal-binding properties of the Cd(4)-containing domain of alpha-rhMT1a assembled into chains of two and three concatenated domains, that is, "necklaces", have been studied by spectrometric techniques, and the interactions within the structures modelled and interpreted by using molecular dynamics. These chains are metallated with 4, 8 or 12 Cd(II) ions to the 11, 22, and 33 cysteinyl sulfur atoms in the alpha-rhMT1a, alphaalpha-rhMT1a, and alphaalphaalpha-rhMT1a proteins, respectively. The effect of pH on the folding of each protein was studied by ESI-MS and optical spectroscopy. MM3/MD simulations were carried out over a period of up to 500 ps by using force-field parameters based on the reported structural data. These calculations provide novel information about the motion of the clustered metallated, partially demetallated, and metal-free peptide chains, with special interest in the region of the metal-binding site. The MD energy/time trajectory conformations show for the first time the flexibility of the metal-sulfur clusters and the bound amino acid chains. We report unexpected and very different sizes for the metallated and demetallated proteins from the combination of experimental data, with molecular dynamics simulations.     

Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase

The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.     

Crystal–nematic phase separation in an asymmetric liquid crystal dimer possessing a terminal hydroxyl group

We prepared an asymmetric liquid crystal dimer possessing a terminal hydroxyl group, 2-{4-{7-[4-(4-cyanophenyl)phenyloxy]heptyloxy}phenyl}-5-(6-hydroxyhexyloxy)pyrimidine, and investigated the phase transition behaviour using polarising optical microscopy and differential scanning calorimetry. The compound exhibited an enantiotropic nematic phase. On cooling, two distinct domains were formed during the nematic-to-crystal phase transition. During heating, one domain melted at 126.4°C to the N phase, whereas the other domain changed to the N phase at 144.6°C. Therefore, the crystal and nematic phases coexisted at a temperature of 18.2 K. The coexistence behaviour characteristics depend on the cooling rate. We discuss the manner in which intermolecular interactions affect the phase transition behaviour.     

Anisotropic nuclear spin interactions for the morphology analysis of proteins in solution by NMR spectroscopy.

Determining the relative orientation of domains within a protein is an important problem in structural biology, which has been difficult to address by either X-ray crystallography or NMR. The structure of a multidomain protein in a crystal lattice can be altered by crystal packing forces, resulting in different domain arrangements from those in solution. On the other hand, conventional NMR primarily provides short-range structural information, including proton-proton distances derived from nuclear Overhauser effects (NOEs) and torsion angles through vicinal spin couplings. Thus, NMR cannot always determine the precise interdomain arrangements due to the sparsely observed spin interactions between domains. However, the weak alignment of proteins in solution has enabled a new NMR technique to determine the domain arrangement based on the different structural information, which defines the orientation of a structural unit in protein against the magnetic field. This technique relies on the anisotropic nuclear spin interactions that only occur for a molecule in a weakly aligned state. In this review, the basics of the new NMR approach are described with focusing on its application to domain orientation analysis. We also describe our recently established NMR approach using the same spin interactions, which expands the domain arrangement analysis to higher-molecular weight proteins over 100 kDa.     

Prediction of number and position of domain boundaries in multi-domain proteins by use of amino acid sequence alone

Prediction of protein domain boundaries is an important step for the prediction of three-dimensional structure. The simple method PDP has been elaborated for prediction of the number and position of domain boundaries in multi-domain proteins by use of amino acid sequence alone. The method uses an optimized scale based on the statistics of appearance of amino acid residues at domain boundaries. Our method demonstrates promising results in comparison to other methods that do not use homologous sequences. From the database of proteins that are targets from CASP6 (Critical Assessment of Techniques for Protein Structure Prediction) our program correctly assigned the number of domains for approximately 80% of one domain proteins and approximately 50% for two-domain proteins. Our method offers three main advantages: it is very simple, it is fast, and it uses a minimal number of parameters in comparison with other methods.     

Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides

BACKGROUND: Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. RESULTS: The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. CONCLUSIONS: A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.     

Domain boundary prediction based on profile domain linker propensity index

Successful prediction of protein domain boundaries provides valuable information not only for the computational structure prediction of multi-domain proteins but also for the experimental structure determination. In this work, a novel index at the profile level is presented, namely, the profile domain linker propensity index (PDLI), which uses the evolutionary information of profiles for domain linker prediction. The frequency profiles are directly calculated from the multiple sequence alignments outputted by PSI-BLAST and converted into binary profiles with a probability threshold. PDLI is then obtained by the frequencies of binary profiles in domain linkers as compared to those in domains. A smooth and normalized numeric profile is generated for any amino acid sequences from which the domain linkers can be predicted. Testing on the Structural Classification of Proteins (SCOP) database and CASP6 targets shows that PDLI outperforms other indexes at the amino acid level.     

Decoding the components of dynamics in three‐domain proteins

In this study, we examine the feasibility and limitations of describing the motional behavior of three‐domain proteins in which the domains are linearly connected. In addition to attempting the determination of the internal and overall reorientational correlation times, we investigate the existence of correlations in the motions between the three domains. Since in linearly arranged three‐domain proteins, there are typically no experimental data that can directly report on motional correlation between the first and the third domain, we address this question by dynamics simulations. Two limiting cases occur: (1) for weak repulsive potentials and (2) when strong repulsive potentials are applied between sequential domains. The motions of the terminal domains become correlated in the case of strong interdomain repulsive potentials when these potentials do not allow the angle between the sequential domains to be smaller than about 60°. Using the model‐free (MF) and extended MF formalisms of Lipari and Szabo, we find that the motional behavior can be separated into two components; the first component represents the concerted overall motion of the three domains, and the second describes the independent component of the motion of each individual domain. We find that this division of the motional behavior of the protein is maintained only when their timescales are distinct and can be made when the angles between sequential domains remain between 60° and 160°. In this work, we identify and quantify interdomain motional correlations. © 2013 Wiley Periodicals, Inc.     

Design of two-dimensional photonic crystal biosensor using DNA detection

Abstract

In this paper, the photonic crystal biosensor is investigated theoretically with, concomitantly, high quality factor, transmission and sensitivity. This biosensor is made out of two waveguide couplers and one L2 resonant cavity formed by removing two air holes. For biosensor analysis, the 2D finite difference time domain (FDTD) method and the plane-wave expansion (PWE) approach are applied. Four slots placed into the cavity and the three rows of functionalized holes nearby the resonant cavities are filled with DNA. For the optimized structure, the biosensor quality factor is found to be over 3.7468?×?10⁶ and the sensitivity is of order 460?nm/RIU. The designed structure has high sensitivity, which is an important parameter in biosensing applications.     

Efficient Multiple Domain Ligation for Proteins Using Asparaginyl Endopeptidase by Selection of Appropriate Ligation Sites Based on Steric Hindrance

Three domain fragments of a multi-domain protein, ER-60, were ligated in two short linker regions using asparaginyl endopeptidase not involving denaturation. To identify appropriate ligation sites, by selecting several potential ligation sites with fewer mutations around two short linker regions, their ligation efficiencies and the functions of the ligated ER-60s were examined experimentally. To evaluate the dependence of ligation efficiencies on the ligation sites computationally, steric hinderances around the sites for the ligation were calculated through molecular dynamics simulations. Utilizing the steric hindrance, a site-dependent ligation potential index was introduced as reproducing the experimental ligation efficiency. Referring to this index, the reconstruction of ER-60 was succeeded by the ligation of the three domains for the first time. In addition, the new ligation potential index well-worked for application to other domain ligations. Therefore, the index may serve as a more time-effective tool for multi-site ligations.     

Molecular dynamics simulation of the LOV2 domain from Adiantum capillus-veneris

The mechanism for signal transduction from the LOV-domains toward the kinase region of phototropin is still not well understood. We have performed molecular dynamics (MD) simulations and CONCOORD calculations on the LOV2 domain of Adiantum capillus-veneris, with the goal to detect possible differences between the two forms of the LOV domain which may not show up in the static crystal structures. Since no such clear differences are found in the MD simulations also, we suggest that the real, biologically active conformation of the LOV domain within the whole phototropin is different from the crystal structure of the isolated LOV domains. The MD simulations do offer, however, insight into details of the dynamics of the dark and illuminated LOV domains, which are discussed in the light of recent experiments.     

From the X-ray compact structure to the elongated form of the full-length MMP-2 enzyme in solution: a molecular dynamics study

Current understanding on the collagenolytic activity performed by the MMPs assumes some degree of relative motion between the catalytic and the hemopexin-like domains of the enzyme. However, all the crystal structures available for the full-length enzymes display a compact arrangement of the protein domains. Herein, we employ Molecular Dynamics simulations to investigate the structure of the full-length MMP-2 enzyme in aqueous solution. This simulation, together with previous experimental results that have been obtained very recently for the MMP-9 and MMP-12 enzymes, gives strong support to the hypothesis that the interdomain dynamics of the MMP enzymes in solution can result in a manifold of conformations including some structures with a large interdomain separation. The simulation of MMP-2 provides also a detailed molecular picture of the structures involved in the transition from the compact X-ray arrangement to the extended form in solution. Such information could be helpful in future studies of the regulation and/or the collagenolytic activity of these important enzymes.     

Investigating chelating sulfonamides and their use in metalloproteinase inhibitors

Matrix metalloproteinase inhibitors (MMPi) utilize zinc-binding groups (ZBGs) to chelate the catalytic Zn(II) ion resulting in enzyme inhibition. Adapting findings from the literature of Zn(II) ion sensors, we previously reported chelating sulfonamide inhibitors of MMP-2, some of which showed excellent selectivity over other gelatinases (MMP-9). Herein, we greatly expand our investigation of chelating sulfonamides as MMP inhibitors (MMPi) with the synthesis and screening of several new libraries consisting of 2-phenyl-7-sulfonamidobenzimidazole, 2-phenyl-7-sulfonamidobenzoxazole, 7-sulfonamidobenzimidazole, 7-sulfonamidobenzoxazole, and 2-(2-sulfonamidophenyl)-quinoline ZBG derivatives. A novel microwave irradiation synthetic procedure was utilized to rapidly and efficiently prepare these molecules. To better understand the coordination chemistry underlying these ZBGs, crystal structures of representative molecules with several first row transition metals were determined and differences in coordination preferences were considered. Surprisingly, only compounds with the 2-phenyl-7-sulfonamidobenzimidazole ZBG showed inhibition of MMP-2, suggesting that the specific structure of the ZBG can have a pronounced effect of inhibitory activity.