scholarly journals Stability and dynamics interrelations in a Lipase: Mutational and MD simulations based investigations

2019 ◽  
Author(s):  
Tushar Ranjan Moharana ◽  
Virendra Kumar ◽  
N. Madhusudhana Rao
Keyword(s):  
Amino Acid ◽  
Outer Region ◽  
Md Simulations ◽  
Parameter Analysis ◽  
Amide Bond ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Amino Acid Region ◽  
Non Local ◽  
Acid Region

AbstractDynamics plays crucial role in the function and stability of proteins. Earlier studies have provided ambivalent nature of these interrelations. Epistatic effects of amino acid substitutions on dynamics are an interesting strategy to investigate such relations. In this study we investigated the interrelation between dynamics with that of stability and activity ofBacillus subtilislipase (BSL) using experimental and molecular dynamics simulation (MDS) approaches. Earlier we have identified many stabilising mutations in BSL using directed evolution. In this study these stabilizing mutations were clustered based on their proximity in the sequence into four groups (CM1 to 4). Activity, thermal stability, protease stability and aggregations studies were performed on these four mutants, along with the wild type BSL, to conclude that the mutations in each region contributed additively to the overall stability of the enzyme without suppressing the activity. Root mean square fluctuation and amide bond squared order parameter analysis from MDS revealed that dynamics has increased for CM1, CM2 and CM3 compared to the wild type in the amino acid region 105 to 112 and for CM4 in the amino acid region 22 to 30. In all the mutants core regions dynamics remained unaltered, while the dynamics in the rigid outer region (RMSF <0.05 nm) has increased. Alteration in dynamics, took place both in the vicinity (CM2, 0.41 nm) as well as far away from the mutations (CM1, 2.6 nm; CM3 1.5 nm; CM4 1.7 nm). Our data suggests that enhanced dynamics in certain regions in a protein may actually improve stability.Statement of SignificanceHow does a protein readjust its dynamics upon incorporation of an amino acid that improved its stability? Are the stabilizing effects of a substitution being local or non-local in nature? While there is an excellent documentation (from x-ray studies) of both local and non-local adjustments in interactions upon incorporation of a stabilizing mutations, the effect of these on the protein dynamics is less investigated. The stability and MD data presented here on four mutants, stabilized around four loop regions of a lipase, suggests that stabilizing effects of these mutations influence two specific regions leaving rest of the protein unperturbed. In addition, our data supports, observations by others, wherein enhancement in stability in a protein need not result in dampening of dynamics of a protein.

The Regulatory Function Of Amino Acid Region 473-487 Of Prothrombin During Coagulation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3574-3574
Author(s):  
Joesph R Wiencek ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Fold Increase ◽  
The United States ◽  
Regulatory Function ◽  
Wild Type ◽  
Amino Acid Region ◽  
Initial Cleavage ◽  
Acid Region ◽  
Thrombin Formation

Abstract Background In the United States, every thirty nine seconds an individual dies from complications from Cardiovascular Diseases. Persistent thrombus formation at the genesis of these diseases, such as stroke and other coagulation disorders, has no full model to date. Intrinsically blood clots are produced due to excessive/unnecessary thrombin formation, which leads to the conversion of fibrinogen to fibrin. As a result the regulation of thrombin formation is critical in controlling clot generation. Upon vasculature damage, the proteolytic conversion of prothrombin (Pro) to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of calcium ions. Although fXa is capable of activating Pro through the initial cleavage at Arg271 followed by the cleavage at Arg320 (pre2 pathway), it would take approximately six months to form a clot. However, the incorporation of fVa into prothrombinase results in a five-fold increase in the catalytic efficiency of fXa for thrombin generation and the order of cleavages reversed (meizo pathway). Thus the timely arrest of unwarranted bleeding is due to the assembly of prothrombinase at the site of injury. Inevitably the presence and absence of fVa dictates the pathway of Pro activation and previous studies have suggested that fXa interacts with Pro within amino acid region 473-487 in a fVa-dependent manner. Aim To evaluate the role amino acid region 473-487 of Pro has in coagulation. Methods A recombinant Pro molecule with the region 473-487 was deleted (rProΔ473-487) using site-directed mutagenesis. Methotrexate was used for selection to stably transfect BHK-21 cells with rProΔ473-487 and wild-type Pro (rProWT). The two recombinant molecules were purified according to a well-established protocol and, at the last step, Fast Performance Liquid Chromatography was used equipped with a strong anionic Mono-Q 5/50 column. Properly carboxylated rProΔ473-487 and rProWT was isolated and removed from the column by utilizing a calcium gradient. Subsequently Pro deficient plasma was used to assess the molecules clotting activities on a Diagnostica Stago STart® 4 Hemostasis Analyzer. Gel electrophoresis was used to evaluate both recombinant molecules and their ability to generate active thrombin by either the multifaceted prothrombinase or fXa alone. Further studies were then performed using generated recombinant thrombin from the recombinant Pro molecules to investigate in their ability to activate procofactors V (fV) & VIII (fVIII). Results The investigation into the Activated Partial Thromboplastin Time [APTT] revealed clotting activity for human Pro and rProWT to be comparable, whereas rProΔ473-487 was substantially limited in the process of forming a fibrin clot. Next gel electrophoresis and scanning densitometry indicated the consumption of rProΔ473-487 by prothrombinase and subsequent thrombin formation was decreased 24-fold when compared to rProWT. In contrast membrane-bound fXa alone, in the absence of fVa, exhibited a 6-fold increase in the rate of initial cleavage Arg271 and subsequent activation of rProΔ473-487. Both recombinant Pro molecules demonstrated a similar cleavage pattern of activation equivalent with human Pro suggesting no structural alterations took place in rProΔ473-487following the mutation. Furthermore, generated human thrombin and recombinant wild-type thrombin were found to activate fV and fVIII within five minutes while the recombinant mutant thrombin was impeded in the activation process out to three hours. Conclusion Overall the data demonstrate that amino acid sequence 473-487 of Pro plays a preeminent role in 1) timely activation of Pro at initial cleavage Arg320 by prothrombinase, and 2) suitable macromolecular procofactor activation. Thus there is incisive rationale why no major mutations have been identified in this dynamic region which would be problematic for inherent physiological hemostasis. Disclosures: No relevant conflicts of interest to declare.

Identification of Sulfo-Tyrosines of Factor V.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1731-1731
Author(s):  
Evrim Erdogan ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Factor Xa ◽  
Factor V ◽  
Wild Type ◽  
Single Chain ◽  
Amino Acid Region ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Cofactor Activity ◽  
Acid Region

Abstract The factor Va molecule is the essential cofactor of the prothrombinase complex. This complex composed of factor Xa and factor Va assembled on a platelet membrane-surface in the presence of Ca2+ ions converts membrane-bound prothrombin to thrombin. Single chain factor V does not bind factor Xa. Single-chain factor V is cleaved by thrombin first at Arg709 followed by cleavages at Arg1018 and Arg1545 to produce the heavy and light chains of the active cofactor (factor Va) and two activation peptides. Efficient thrombin cleavage and activation of factor V is essential for cofactor function and requires tyrosine sulfation. Tyrosine sulfation of factor V also appears to regulate its activity. Seven tyrosine residues in factor V, Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, and Tyr1565 have been identified as potential sites of sulfation. However, which residues are sulfated and their contribution to procofactor activation and cofactor function still remain to be elucidated. Two of the sulfation sites Tyr696 and Tyr698 are located in the acidic amino acid region near to the first required thrombin cleavage site at Arg709. Recent data demonstrated that these residues are essential for factor V activation and cofactor activity. Another acidic amino acid region, 1490–1520 is adjacent to the thrombin cleavage site at Arg1545 required for light chain formation. This region also contains three potential sulfation sites at residues 1494, 1510, and 1515 and was shown to be required for optimum procofactor activation. To ascertain which of these three residues is important for procofactor activation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations 1493DY1494→AF, 1508DDY1510→AAF and 1514DY1515→AF. The clotting and cofactor activity of the 1493DY1494→AF and 1514DY1515→AF mutants was similar to the clotting activity observed with the wild type recombinant factor Va molecule following activation by thrombin or RVV-V activator. In contrast, under similar experimental conditions recombinant factor V with the substitution 1508DDY1510→AAF was deficient in its clotting activity and had impaired cofactor activity. Moreover, following prolonged incubation with thrombin, no light chain formation was observed in the factor V molecule bearing the 1508DDY1510→AAF mutation. Thus, amino acid residues 1508–1510 of factor V are required for thrombin interaction with the procofactor which in turn appears necessary for cleavage at Arg1545. Studies of sulfated proteins have shown that the effect of sulfo-tyrosines on protein structure/function can be preserved by replacing them with glutamic acid. To explicitly identify the sulfated tyrosines on the factor V molecule, we mutated Tyr696, Tyr698 and Tyr1510 to glutamic acid and transfected them into COS-7L cells. Expression was performed in the presence of media containing or devoid of sulfate. In the presence of sulfate, the cofactor and clotting activities of the DY696DY698→DEDE and DDY1510→DDE mutants, separately were similar to the wild type recombinant factor Va molecule. However, in the absence of sulfate, the wild type and the mutant recombinant factor V molecules had both impaired cofactor activity and clotting activity following their activation with thrombin. However, their respective activity was higher than the activity of the factor V molecule bearing the 1508DDY1510→AAF mutation. Our data suggest that residues 696, 698, and 1510 of factor V appear to be sulfated and might be important for procofactor activation and cofactor function.

Wingless signaling generates pattern through two distinct mechanisms

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3727-3736 ◽  
Author(s):  
R. Hays ◽  
G.B. Gibori ◽  
A. Bejsovec
Keyword(s):  
Cell Fate ◽  
Cellular Response ◽  
Biological Activities ◽  
Essential Feature ◽  
Structural Features ◽  
Protein Distribution ◽  
Wild Type ◽  
Cell Fates ◽  
Amino Acid Region ◽  
Acid Region

wingless (wg) and its vertebrate homologues, the Wnt genes, play critical roles in the generation of embryonic pattern. In the developing Drosophila epidermis, wg is expressed in a single row of cells in each segment, but it influences cell identities in all rows of epidermal cells in the 10- to 12-cell-wide segment. Wg signaling promotes specification of two distinct aspects of the wild-type intrasegmental pattern: the diversity of denticle types present in the anterior denticle belt and the smooth or naked cuticle constituting the posterior surface of the segment. We have manipulated the expression of wild-type and mutant wg transgenes to explore the mechanism by which a single secreted signaling molecule can promote these distinctly different cell fates. We present evidence consistent with the idea that naked cuticle cell fate is specified by a cellular pathway distinct from the denticle diversity-generating pathway. Since these pathways are differentially activated by mutant Wg ligands, we propose that at least two discrete classes of receptor for Wg may exist, each transducing a different cellular response. We also find that broad Wg protein distribution across many cell diameters is required for the generation of denticle diversity, suggesting that intercellular transport of the Wg protein is an essential feature of pattern formation within the epidermal epithelium. Finally, we demonstrate that an 85 amino acid region not conserved in vertebrate Wnts is dispensable for Wg function and we discuss structural features of the Wingless protein required for its distinct biological activities.

scholarly journals A Carboxy-Terminal 16-Amino-Acid Region of ς38 ofEscherichia coli Is Important for Transcription under High-Salt Conditions and Sigma Activities In Vivo

2000 ◽  
Vol 182 (16) ◽  
pp. 4628-4631 ◽  
Author(s):  
Mio Ohnuma ◽  
Nobuyuki Fujita ◽  
Akira Ishihama ◽  
Kan Tanaka ◽  
Hideo Takahashi
Keyword(s):  
Amino Acid ◽  
Bacterial Species ◽  
High Potassium ◽  
Transcription Analysis ◽  
Amino Acid Region ◽  
Sigma Subunit ◽  
Carboxy Terminal ◽  
Acid Region

ABSTRACT ς38 (or ςS, the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of ς38 (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant ς38 retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of ς38 holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.

scholarly journals The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix.

1994 ◽  
Vol 14 (4) ◽  
pp. 2755-2766 ◽  
Author(s):  
D G Overdier ◽  
A Porcella ◽  
R H Costa
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Hepatocyte Nuclear Factor ◽  
Binding Properties ◽  
Winged Helix ◽  
Amino Acid Region ◽  
Dna Binding Specificity ◽  
Recognition Helix ◽  
Dna Binding Properties ◽  
Acid Region

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.

scholarly journals Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton inSaccharomyces cerevisiae

1998 ◽  
Vol 9 (5) ◽  
pp. 1221-1233 ◽  
Author(s):  
Takeshi Fujiwara ◽  
Kazuma Tanaka ◽  
Akihisa Mino ◽  
Mitsuhiro Kikyo ◽  
Kazuo Takahashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Microscopic Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Gene Encoding ◽  
Amino Acid Region ◽  
Genes Encoding ◽  
Mitogen Activated Protein ◽  
Acid Region

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both thebni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

The F12-Vif derivative Chim3 inhibits HIV-1 replication in CD4+ T lymphocytes and CD34+-derived macrophages by blocking HIV-1 DNA integration

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3443-3452 ◽  
Author(s):  
Simona Porcellini ◽  
Luca Alberici ◽  
Francesco Gubinelli ◽  
Rossella Lupo ◽  
Clelia Olgiati ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
T Lymphocytes ◽  
Genetic Manipulation ◽  
Natural Resistance ◽  
Dna Integration ◽  
Amino Acid Region ◽  
Antiviral Function ◽  
Hiv 1 ◽  
Acid Region

Abstract The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti–HIV-1/AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4+ T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34+ human stem cells (HSCs). Moreover, we identified the 126- to 170-amino-acid region in the C-terminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. First, Chim3 functions as a transdominant factor that preserves the antiviral function of the natural restriction factor APOBEC3G (hA3G). Second, Chim3 blocks the early HIV-1 retrotranscript accumulation and thereby HIV-1 DNA integration regardless of the presence of WT-Vif and hA3G. In conclusion, by impairing the early steps of HIV-1 life cycle, Chim3 conceivably endows engineered cells with survival advantage, which is required for the efficient immune reconstitution of patients living with HIV/AIDS.

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