scholarly journals Reactive Centre Loop Dynamics and Serpin Specificity

2018 ◽  
Author(s):  
Emilia M. Marijanovic ◽  
James Fodor ◽  
Blake T. Riley ◽  
Benjamin T. Porebski ◽  
Mauricio G. S. Costa ◽  
...  
Keyword(s):  
Proteinase Inhibitors ◽  
Conformational Dynamics ◽  
Serine Proteinase ◽  
Human Neutrophil Elastase ◽  
Protease Inhibition ◽  
Reactive Centre Loop ◽  
Substrate Engineering ◽  
Model Protein ◽  
Dynamics Simulations ◽  
And Function

AbstractSerine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as a1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

scholarly journals Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Stephan Hirschi ◽  
David Kalbermatter ◽  
Zöhre Ucurum ◽  
Thomas Lemmin ◽  
Dimitrios Fotiadis
Keyword(s):  
Proton Translocation ◽  
Functional Characterization ◽  
Green Light ◽  
Proton Donor ◽  
Molecular Organization ◽  
Primary Proton ◽  
Proton Pumps ◽  
Model Protein ◽  
Dynamics Simulations ◽  
And Function

AbstractThe green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

Structure and function of invertebrate Kazal-type serine proteinase inhibitors

2010 ◽  
Vol 34 (4) ◽  
pp. 377-386 ◽  
Author(s):  
Vichien Rimphanitchayakit ◽  
Anchalee Tassanakajon
Keyword(s):  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Structure And Function ◽  
Serine Proteinase Inhibitors ◽  
And Function

scholarly journals Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

2020 ◽  
Author(s):  
Matthias Fellner ◽  
Rinky Parakra ◽  
Kirstin O. McDonald ◽  
Itamar Kass ◽  
Guy N.L. Jameson ◽  
...  
Keyword(s):  
Peroxidase Activity ◽  
Conformational Dynamics ◽  
Cellular Processes ◽  
Pathogenic Variants ◽  
Alkaline Transition ◽  
Apoptotic Activity ◽  
Dynamics Simulations ◽  
And Function ◽  
Platelet Formation

AbstractMutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with exchange between conformational states proposed to regulate changes in function. Here we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. We find that three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with increased overall flexibility of cytochrome c, particularly movement of the Ω loops. This suggests that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an “induced fit” mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics suggesting that the mechanism by which the variants alter peroxidase activity is not related to the conformation dynamics of the hexacoordinate heme Fe state of cytochrome c analyzed in the simulations. Recent suggestions that conformational mobility of specific regions of cytochrome c underpins changes in reduction potential and the alkaline transition pK were not supported. These data highlight that conformational dynamics of cytochrome c drives some but not all of its properties and activities.

scholarly journals Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer

2017 ◽  
Vol 114 (10) ◽  
pp. E1786-E1795 ◽  
Author(s):  
Suraj Adhikary ◽  
Daniel J. Deredge ◽  
Anu Nagarajan ◽  
Lucy R. Forrest ◽  
Patrick L. Wintrode ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Conformational Dynamics ◽  
Complete Removal ◽  
Synaptic Cleft ◽  
Binding Activity ◽  
Phospholipid Bilayer ◽  
X Ray Crystallography ◽  
Model Protein ◽  
Clinically Significant ◽  
Dynamics Simulations

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.

scholarly journals Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

2021 ◽  
Vol 478 (3) ◽  
pp. 669-684
Author(s):  
Matthias Fellner ◽  
Rinky Parakra ◽  
Kirstin O. McDonald ◽  
Itamar Kass ◽  
Guy N.L. Jameson ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Cytochrome C ◽  
Peroxidase Activity ◽  
Conformational Dynamics ◽  
Cellular Processes ◽  
Pathogenic Variants ◽  
Alkaline Transition ◽  
Apoptotic Activity ◽  
Dynamics Simulations ◽  
And Function

Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an ‘induced fit’ mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.

A Novel Serpin Expressed by Blood-Borne Microfilariae of the Parasitic Nematode Brugia malayi Inhibits Human Neutrophil Serine Proteinases

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1418-1428 ◽  
Author(s):  
Xingxing Zang ◽  
Maria Yazdanbakhsh ◽  
Haobo Jiang ◽  
Michael R. Kanost ◽  
Rick M. Maizels
Keyword(s):  
Human Neutrophil ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Brugia Malayi ◽  
Cathepsin G ◽  
Cdna Libraries ◽  
Human Neutrophil Elastase ◽  
Serine Proteinases ◽  
Serpin Gene ◽  
Wide Range

Abstract Serine proteinase inhibitors (serpins) play a vital regulatory role in a wide range of biological processes, and serpins from viruses have been implicated in pathogen evasion of the host defence system. For the first time, we report a functional serpin gene from nematodes that may function in this manner. This gene, named Bm-spn-2, has been isolated from the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis. Polymerase chain reaction (PCR) and Western blot experiments indicate that Bm-spn-2 is expressed only by microfilariae (Mf), which are the long-lived blood-dwelling larval stage. A survey of the greater than 14,000 expressed sequence tags (ESTs) from B malayi deposited in dbEST shows that greater than 2% of the ESTs sequenced from Mf cDNA libraries correspond to Bm-spn-2. Despite its abundance in the microfilarial stage, Bm-spn-2 has not been found in any other point in the life cycle. The predicted protein encoded byBm-spn-2 contains 428 amino acids with a putative signal peptide. Antibodies to recombinant Bm-SPN-2 protein react specifically with a 47.5-kD native protein in Mf extract. Bm-SPN-2 is one of the largest of the 93 known serpins, due to a 22 amino acid carboxy-terminal extension, and contains the conserved serpin signature sequence. Outside these regions, levels of homology are low, and only a distant relationship can been seen to a Caenorhabditis elegansserpin. The Bm-spn-2 gene contains 6 introns, 2 of which appear to be shared by both nematode species. The B malayi introns have an extended and conserved 3′ splice site and are relatively large compared with C elegans. A panel of mammalian serine proteinases were screened and Bm-SPN-2 protein was found to specifically inhibit enzymatic activity of human neutrophil cathepsin G and human neutrophil elastase, but not a range of other serine proteinases. It is possible that Bm-SPN-2 could function as a stage-specific serpin in the blood environment of the microfilarial parasite in protection from human immunity and thus may be a good candidate for protective vaccine.

A Novel Serpin Expressed by Blood-Borne Microfilariae of the Parasitic Nematode Brugia malayi Inhibits Human Neutrophil Serine Proteinases

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1418-1428 ◽  
Author(s):  
Xingxing Zang ◽  
Maria Yazdanbakhsh ◽  
Haobo Jiang ◽  
Michael R. Kanost ◽  
Rick M. Maizels
Keyword(s):  
Human Neutrophil ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Brugia Malayi ◽  
Cathepsin G ◽  
Cdna Libraries ◽  
Human Neutrophil Elastase ◽  
Serine Proteinases ◽  
Serpin Gene ◽  
Wide Range

Serine proteinase inhibitors (serpins) play a vital regulatory role in a wide range of biological processes, and serpins from viruses have been implicated in pathogen evasion of the host defence system. For the first time, we report a functional serpin gene from nematodes that may function in this manner. This gene, named Bm-spn-2, has been isolated from the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis. Polymerase chain reaction (PCR) and Western blot experiments indicate that Bm-spn-2 is expressed only by microfilariae (Mf), which are the long-lived blood-dwelling larval stage. A survey of the greater than 14,000 expressed sequence tags (ESTs) from B malayi deposited in dbEST shows that greater than 2% of the ESTs sequenced from Mf cDNA libraries correspond to Bm-spn-2. Despite its abundance in the microfilarial stage, Bm-spn-2 has not been found in any other point in the life cycle. The predicted protein encoded byBm-spn-2 contains 428 amino acids with a putative signal peptide. Antibodies to recombinant Bm-SPN-2 protein react specifically with a 47.5-kD native protein in Mf extract. Bm-SPN-2 is one of the largest of the 93 known serpins, due to a 22 amino acid carboxy-terminal extension, and contains the conserved serpin signature sequence. Outside these regions, levels of homology are low, and only a distant relationship can been seen to a Caenorhabditis elegansserpin. The Bm-spn-2 gene contains 6 introns, 2 of which appear to be shared by both nematode species. The B malayi introns have an extended and conserved 3′ splice site and are relatively large compared with C elegans. A panel of mammalian serine proteinases were screened and Bm-SPN-2 protein was found to specifically inhibit enzymatic activity of human neutrophil cathepsin G and human neutrophil elastase, but not a range of other serine proteinases. It is possible that Bm-SPN-2 could function as a stage-specific serpin in the blood environment of the microfilarial parasite in protection from human immunity and thus may be a good candidate for protective vaccine.

Plasminogen Activator and Plasminogen Activator Inhibitor Associated with Granulomatous Inflammation: A Study with Murine Leprosy

1984 ◽  
Vol 52 (03) ◽  
pp. 243-249 ◽  
Author(s):  
S Izaki ◽  
T Hibino ◽  
Y Isozaki ◽  
P S Hsu ◽  
M Izaki ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Soluble Fraction ◽  
Proteinase Inhibitors ◽  
Granulomatous Inflammation ◽  
Serine Proteinase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Weakly Alkaline ◽  
Heat Stable ◽  
Type Plasminogen Activator

SummaryPlasminogen activator that is associated with the development of hypersensitivity granulomas (gPA) was partially purified from a saline soluble fraction of murine lepromas elicited in “resistant” mice, C57BL/6N. The gPA was shown to consist of two subspecies (23,000 and 48,000 in molecular weight) with essentially identical enzymologic properties. The gPA was found to be a relatively heat stable weakly alkaline serine proteinase with trypsin-like characteristics in the specificity for synthetic substrates and proteinase inhibitors. It showed a high affinity for H- D-Ile-Pro-Arg-pNA (Km = 1.4 × 10-4 M) H-D-Val-Leu-Lys- pNA (Km = 5.2 × 10-4 M), and L-pyroGlu-Gly-Arg-pNA (Km = 9.3 × 10-4 M). The gPA did not demonstrate antigenic cross reaction with urokinase-type or tissue-type plasminogen activator.Two distinct enzymatic regulators of the gPA were also demonstrated in the saline soluble fraction of the hypersensitivity granulomas. The gPA and its regulation are assumed to be correlated with macrophage activation in the hypersensitivity granulomas

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection

2020 ◽  
Vol 16 (4) ◽  
pp. 451-459 ◽  
Author(s):  
Fortunatus C. Ezebuo ◽  
Ikemefuna C. Uzochukwu
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Binding Energy ◽  
Binding Site ◽  
Conformational Dynamics ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Conformational Selection ◽  
Hybrid Mechanism ◽  
Dynamics Simulations

Background: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. Objective: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Methods: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it’s missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. Results: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. Conclusion: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

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