scholarly journals Identification of furin pro-region determinants involved in folding and activation

2004 ◽  
Vol 379 (3) ◽  
pp. 757-763 ◽  
Author(s):  
Lyne BISSONNETTE ◽  
Gabriel CHAREST ◽  
Jean-Michel LONGPRÉ ◽  
Pierre LAVIGNE ◽  
Richard LEDUC
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Hydrophobic Core ◽  
Binding Interface ◽  
Golgi Network ◽  
Von Willebrand ◽  
Pro Region

The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the trans-Golgi network. To identify residues that are important for pro-region function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furin's pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furin's pro-region.

scholarly journals The Amino Terminus of Varicella-Zoster Virus (VZV) Glycoprotein E Is Required for Binding to Insulin-Degrading Enzyme, a VZV Receptor

2007 ◽  
Vol 81 (16) ◽  
pp. 8525-8532 ◽  
Author(s):  
Qingxue Li ◽  
Tammy Krogmann ◽  
Mir A. Ali ◽  
Wei-Jen Tang ◽  
Jeffrey I. Cohen
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Dependent Manner ◽  
Insulin Degrading Enzyme ◽  
Concentration Dependent Manner ◽  
Glycoprotein E ◽  
Degrading Enzyme ◽  
Varicella Zoster

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is required for VZV infection. Although gE is well conserved among alphaherpesviruses, the amino terminus of VZV gE is unique. Previously, we showed that gE interacts with insulin-degrading enzyme (IDE) and facilitates VZV infection and cell-to-cell spread of the virus. Here we define the region of VZV gE required to bind IDE. Deletion of amino acids 32 to 71 of gE, located immediately after the predicted signal peptide, resulted in loss of the ability of gE to bind IDE. A synthetic peptide corresponding to amino acids 24 to 50 of gE blocked its interaction with IDE in a concentration-dependent manner. However, a chimeric gE in which amino acids 1 to 71 of VZV gE were fused to amino acids 30 to 545 of herpes simplex virus type 2 gE did not show an increased level of binding to IDE compared with that of full-length HSV gE. Thus, amino acids 24 to 71 of gE are required for IDE binding, and the secondary structure of gE is critical for the interaction. VZV gE also forms a heterodimer with glycoprotein gI. Deletion of amino acids 163 to 208 of gE severely reduced its ability to form a complex with gI. The amino portion of IDE, as well an IDE mutant in the catalytic domain of the protein, bound to gE. Therefore, distinct motifs of VZV gE are important for binding to IDE or to gI.

scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

scholarly journals The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex.

1997 ◽  
Vol 17 (2) ◽  
pp. 571-583 ◽  
Author(s):  
F Liu ◽  
J J Stanton ◽  
Z Wu ◽  
H Piwnica-Worms
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Catalytic Domain ◽  
Cyclin B ◽  
Dependent Manner ◽  
Cdc2 Kinase ◽  
Dual Specificity ◽  
Catalytically Active ◽  
Specificity Protein

Entry into mitosis requires the activity of the Cdc2 kinase. Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation. Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory. Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified. Here we report the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner. Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis. Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells. A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, as its deletion results in the localization of Myt1Hu primarily to the nucleus.

scholarly journals Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence

2003 ◽  
Vol 373 (2) ◽  
pp. 571-581 ◽  
Author(s):  
Angie F. KIRWAN ◽  
Ashley C. BIBBY ◽  
Thierry MVILONGO ◽  
Heimo RIEDEL ◽  
Thomas BURKE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Regulatory Domain ◽  
Direct Role ◽  
Independent Manner ◽  
Inhibitory Capacity ◽  
Pseudosubstrate Sequence

The N-terminal pseudosubstrate site within the protein kinase Cα (PKCα)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCα-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940–8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCα-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Rα39–177: this protein contained the full regulatory domain of human PKCα fused to glutathione S-transferase (GST), but lacked amino acids 1–38 (including the pseudosubstrate sequence) and amino acids 178–270 (including the C2 region)]. GST-Rα39–177 significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCα holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33–86 within GST-Rα39–177 dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159–242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCα-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

scholarly journals On the substrate recognition and negative regulation of SPAK, a kinase modulating Na+-K+-2Cl− cotransport activity

2010 ◽  
Vol 299 (3) ◽  
pp. C614-C620 ◽  
Author(s):  
Kenneth B. Gagnon ◽  
Eric Delpire
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Catalytic Domain ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Threonine Residue ◽  
In Vivo Experiments ◽  
Constitutively Active

Threonines targeted by Ste20-related proline-alanine-rich kinase (SPAK) for phosphorylation have been identified in Na+-K+-2Cl− cotransporter type 1 (NKCC1), NKCC2, and Na+-Cl− cotransporter (NCC). However, what constitutes the substrate recognition of the kinase is still unknown. Using site-directed mutagenesis and functional measurement of NKCC1 activity in Xenopus laevis oocytes, we determined that SPAK recognizes two threonine residues separated by four amino acids. Addition or removal of a single residue abrogated SPAK activation of NKCC1. Although both threonines are followed by hydrophobic residues, in vivo experiments have determined that SPAK activation of the cotransporter only requires a hydrophobic residue after the first threonine. Interestingly, downstream of the second threonine residue, we have identified a conserved aspartic acid residue which is critical for NKCC1 function. Mouse SPAK activity requires phosphorylation of two specific residues by WNK [with no lysine (K)] kinases: a threonine (T243) in the catalytic domain and a serine (S383) in the regulatory domain. We found that mutating the threonine residue into a glutamic acid (T243E) combined with mutation of the serine into an aspartic acid (S383D) rendered SPAK constitutively active. Surprisingly, alanine substitution of S383 or mutation of residues surrounding this residue also resulted in a constitutively active kinase. Interestingly, deletion of amino acids 356–398 identified another serine residue in the catalytic domain (S321) as another putative target of WNK phosphorylation. We found that WNK4 is capable of stimulating the deletion mutant when S321 is present, but not when S321 is mutated into an alanine.

scholarly journals Analysis of the Human Interleukin-6/Human Interleukin-6 Receptor Binding Interface at the Amino Acid Level: Proposed Mechanism of Interaction

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1319-1333 ◽  
Author(s):  
Michael Kalai ◽  
Fèlix A. Montero-Julian ◽  
Joachim Grötzinger ◽  
Véronique Fontaine ◽  
Paul Vandenbussche ◽  
...  
Keyword(s):  
Amino Acid ◽  
Interleukin 6 ◽  
Acid Level ◽  
Binding Free Energy ◽  
Contact Region ◽  
Amino Acid Level ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Core ◽  
Α Subunit ◽  
Binding Interface

Abstract The interaction between interleukin-6 (IL-6) and IL-6 receptor (IL-6R) is the initial and most specific step in the IL-6 signaling pathway. Understanding its mechanism at the amino acid level is the basis for developing small IL-6–inhibiting molecules. We studied the human IL-6 (hIL-6)/hIL-6R binding interface by a combination of molecular modelling and site-directed mutagenesis. Our model suggests that the center of the interface between the two molecules consists of hydrophobic contacts predicted to account for most of the binding-free energy. These contacts can be regarded as a hydrophobic core shielded by hydrophilic residues that are also needed for recognition. Following this hypothesis, we altered in hIL-6 and hIL-6R residues predicted to reside in the contact region and to interact with each other. We studied the capacity of these mutants to form an IL-6/IL-6R complex and their ability to transduce the signal. This combined approach has led to the identification of certain residue-clusters in the binding interface and to a rational explanation of their specific interactions, suggesting therein a likely mechanism of complex formation. The results confirm the predictive model and strongly support our hypothesis. Comparison with other cytokines and their α-subunit receptors suggests that the structural location of certain binding sites are conserved.

scholarly journals Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 946-952 ◽  
Author(s):  
DM Haverstick ◽  
JF Cowan ◽  
KM Yamada ◽  
SA Santoro
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Synthetic Peptides ◽  
Dependent Manner ◽  
Cell Binding ◽  
Binding Region ◽  
Concentration Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

The role in platelet function of the cell-binding region of fibronectin was explored by the use of synthetic peptides. The prototypical peptide gly-arg-gly-asp-ser was capable of inhibiting thrombin-induced platelet aggregation without altering the degree of platelet activation as judged by the secretion of 14C-serotonin. The peptide also effectively inhibited, in a concentration-dependent manner, the binding of radiolabeled fibronectin to platelets and the adhesion of platelets to fibronectin substrates. The smallest peptide from the cell-binding region of fibronectin which retained full activity was arg-gly-asp-ser. Transposition of amino acids or conservative substitutions of amino acids within this short sequence resulted in inactive peptides. Peptides containing the arg-gly-asp-ser sequence were also capable of inhibiting the adhesion of platelets to fibrinogen and von Willebrand factor substrates. Examination of the entire panel of synthetic peptides for ability to inhibit adhesion to fibrinogen or von Willebrand factor substrates revealed the same structure-function relationships that had been determined in the studies with fibronectin.

scholarly journals Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 946-952 ◽  
Author(s):  
DM Haverstick ◽  
JF Cowan ◽  
KM Yamada ◽  
SA Santoro
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Synthetic Peptides ◽  
Dependent Manner ◽  
Cell Binding ◽  
Binding Region ◽  
Concentration Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

Abstract The role in platelet function of the cell-binding region of fibronectin was explored by the use of synthetic peptides. The prototypical peptide gly-arg-gly-asp-ser was capable of inhibiting thrombin-induced platelet aggregation without altering the degree of platelet activation as judged by the secretion of 14C-serotonin. The peptide also effectively inhibited, in a concentration-dependent manner, the binding of radiolabeled fibronectin to platelets and the adhesion of platelets to fibronectin substrates. The smallest peptide from the cell-binding region of fibronectin which retained full activity was arg-gly-asp-ser. Transposition of amino acids or conservative substitutions of amino acids within this short sequence resulted in inactive peptides. Peptides containing the arg-gly-asp-ser sequence were also capable of inhibiting the adhesion of platelets to fibrinogen and von Willebrand factor substrates. Examination of the entire panel of synthetic peptides for ability to inhibit adhesion to fibrinogen or von Willebrand factor substrates revealed the same structure-function relationships that had been determined in the studies with fibronectin.

scholarly journals Structure-Function Relationships of the Collagen-Specific Chaperone Hsp47

2014 ◽  
Vol 70 (a1) ◽  
pp. C1631-C1631
Author(s):  
Ulrich Baumann ◽  
Jan Gebauer ◽  
Christine Widmer ◽  
Sinan Oecal ◽  
Frank Zaucke
Keyword(s):  
Binding Sites ◽  
Triple Helix ◽  
Specific Binding ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Missense Mutations ◽  
Ph Change ◽  
Collagen Triple Helix ◽  
Binding Interface ◽  
Cycle Dependent

Hsp47 is an essential collagen specific chaperone that is crucial for proper formation of the collagen triple helix. KO-mice die at an early emybronic state and missense mutations are linked to severe forms of osteogenesis imperfecta (Ishida & Nagata, 2011). On the other hand, Hsp47 is strongly upregulated in fibrotic pathogenies and has in proof-of-principle studies been successfully targeted . It is a very unusual chaperone as it specifically recognises the folded conformation and releases ist client upon changes in pH and not in an ATP-cycle dependent manner. We have deterined the crystal structure of Hsp47 alone and in complex with homotrimeric collagen model peptides (Widmer et al., 2012), revealing a 2:1 stoichiometry Hsp47:collagen-trimer. The specific recognition of an arginine at the Y-position of the triple helix is explained by a saltbridge to an aspartic acid of Hsp47. Based on these structures we have undergone further investigation in order to shed light on the pH-triggered client release and to gain further insight into client recognition. We have undergone a systematic site-directed mutagenesis study of histidine residues in the binding interface. Interestingly, only few side-chains are predicted to change significantly their protonation state by a pH change from 7 to 6. We have furthermore investigated the influence of the reactive center loop, which is disordered in most crystal structures. An open question is how many binding sites for Hsp47 exist in procollagen. EM studies reveal a large number of specific binding sites, explaining how Hsp47 clamps and stabilises the triple helix and prevents lateral aggregation.

scholarly journals Single Amino Acid Change Mutation in the Hydrophobic Core of the N-terminal Domain of P22 TSP affects the Proteins Stability.

2021 ◽  
Author(s):  
Joseph A Ayariga ◽  
Robert Villafane
Keyword(s):  
Amino Acids ◽  
Site Directed Mutagenesis ◽  
Spike Protein ◽  
Single Amino Acid ◽  
In Vitro Assays ◽  
Hydrophobic Core ◽  
Terminal Domain ◽  
Single Amino Acid Change ◽  
The Stability

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly shifted the attention of researchers to critically investigate most viruses to understand specific characteristics that impart their virulence. For instance, the SARS-CoV-2 has undergone several mutations, with some variants classified as variants of concern, e.g., the Omicron and Delta variant of SARS-CoV-2 are known for their rapid transmission and antigenicity due to mutation in the Spike protein. P22 bacteriophage is a bacterial virus that has a tailspike protein (TSP) that performs similar functions as the Spike protein of SARS-COV-2. We previously carried out a site-directed mutagenesis of the P22 TSP to bear disruptive mutations in the hydrophobic core of the N-terminal Domain (NTD), then partially characterized the properties of the mutant TSPs. In this process, the valine patch (triple valine residues that formed a hydrophobic core) was replaced with charged amino acids (Asp or lysine) or hydrophobic amino acids (Leucine or isoleucine). Some of the mutant TSPs characterized showed significant differences in migration in both native and SDS-PAGE. Mutants with such disruptive mutation are known to show non-native properties, and as expected, most of these mutants obtained showed significantly different properties from the WT P22 TSP. In this work, we further characterized these mutant species by computational and in vitro assays to demonstrate the validity of our previous inference that the valine patch is a critical player in the stability of the N-terminal domain of the P22 TSP.

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