scholarly journals The N-Terminal Transmembrane Domain of λ S Is Required for Holin but Not Antiholin Function

2009 ◽  
Vol 192 (3) ◽  
pp. 725-733 ◽  
Author(s):  
Rebecca White ◽  
Tram Anh T. Tran ◽  
Chelsey A. Dankenbring ◽  
John Deaton ◽  
Ry Young
Keyword(s):  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Amino Group ◽  
Missense Mutations ◽  
Wild Type Allele ◽  
Wild Type ◽  
Basic Residue ◽  
N Terminus ◽  
Terminal Amino ◽  
Positively Charged

ABSTRACT The λ S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105ΔTMD1 , with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105ΔTMD1 and holin carried the substitution.

scholarly journals Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Takeshi Nomura ◽  
Masahiro Sokabe ◽  
Kenjiro Yoshimura
Keyword(s):  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Wild Type ◽  
Dependent Inactivation ◽  
Mechanical Threshold ◽  
Voltage Dependent ◽  
Arginine Residues ◽  
Tm Domain ◽  
Small Conductance ◽  
Positively Charged

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure. To examine whether these residues are involved in the voltage dependence of MscS, we neutralized the charge of each residue by substituting with asparagine (R46N, R54N, and R74N). Mechanical threshold for the opening of the expressed wild-type MscS and asparagine mutants did not change with voltage in the range from-40 to +100 mV. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. The wild-type MscS inactivated at +60 to +80 mV but not at-60 to +40 mV. The voltage dependence of the inactivation rate of all mutants tested, that is, R46N, R54N, R74N, and R46N/R74N MscS, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of R46, R54, and R74.

scholarly journals Does Cysteine Rule (CysR) Complete the CendR Principle? Increase in Affinity of Peptide Ligands for NRP-1 Through the Presence of N-Terminal Cysteine

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 448 ◽  
Author(s):  
Anna K. Puszko ◽  
Piotr Sosnowski ◽  
Françoise Raynaud ◽  
Olivier Hermine ◽  
Gérard Hopfgartner ◽  
...  
Keyword(s):  
Peptide Ligands ◽  
Side Chain ◽  
Amino Group ◽  
Structure Activity ◽  
C Terminus ◽  
Neuropilin 1 ◽  
Ic50 Value ◽  
N Terminus ◽  
Terminal Amino ◽  
Relationship Of

The structure-activity relationship of branched H-Lys(hArg)-Dab-Dhp-Arg-OH sequence analogues, modified with Cys-Asp or Cys at N-terminal amino acids (Lys, hArg), in VEGF-A165/Neuropilin-1 complex inhibition is presented. The addition of Cys residue led to a 100-fold decrease in the IC50 value, compared to the parent peptide. The change occurred regardless of coupling Cys to the free N-terminal amino group present in the main or the side chain. A few analogues extended by the attachment of Cys at the N-terminus of several potent NRP-1 peptide ligands documented in the literature are also presented. In all studied cases, the enhancement of inhibitory properties after the addition of Cys at the N-terminus is observed. It is particularly evident for the tetrapeptide derived from the C-terminus of VEGF-A165 (KPRR), suggesting that extending the K/RXXK/R motif (CendR) with the Cys moiety can significantly improve affinity to NRP-1 of CendR peptides.

scholarly journals His20 Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain

2007 ◽  
Vol 189 (7) ◽  
pp. 2825-2833 ◽  
Author(s):  
Ray A. Larsen ◽  
Gail E. Deckert ◽  
Kyle A. Kastead ◽  
Surendranathan Devanathan ◽  
Kimberly L. Keller ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Protonmotive Force ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
In The Wild ◽  
Mutant Phenotypes

ABSTRACT The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His20 is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser16 and His20, the resultant “all-Ala Ser16 His20” TMD TonB retained 90% of wild-type iron transport activity. Ser16Ala in the context of a wild-type TonB TMD was fully active. In contrast, His20Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser16 His20 TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser16 His20 TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser16 His20 TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser16 His20 TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.

scholarly journals Particle Polymorphism Caused by Deletion of a Peptide Molecular Switch in a Quasiequivalent Icosahedral Virus

1998 ◽  
Vol 72 (7) ◽  
pp. 6024-6033 ◽  
Author(s):  
X. Fan Dong ◽  
Padmaja Natarajan ◽  
Mariana Tihova ◽  
John E. Johnson ◽  
Anette Schneemann
Keyword(s):  
Coat Protein ◽  
Single Type ◽  
Flock House Virus ◽  
Wild Type ◽  
Protein Subunits ◽  
Charged Amino Acids ◽  
Irregular Structures ◽  
Icosahedral Virus ◽  
N Terminus ◽  
Positively Charged

ABSTRACT The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.

scholarly journals A P425R mutation of the proton-coupled folate transporter causing hereditary folate malabsorption produces a highly selective alteration in folate binding

2012 ◽  
Vol 302 (9) ◽  
pp. C1405-C1412 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Rongbao Zhao ◽  
Enghui H. Yap ◽  
Andras Fiser ◽  
I. David Goldman
Keyword(s):  
Secondary Structure ◽  
Transmembrane Domain ◽  
Water Soluble ◽  
Wild Type ◽  
Sulfhydryl Reagents ◽  
Functional Changes ◽  
External Loop ◽  
Human Solid Tumors ◽  
Translocation Pathway ◽  
Positively Charged

Proton-coupled folate transporter (PCFT) mediates folate intestinal absorption and transport across the choroid plexus, processes defective in subjects with hereditary folate malabsorption (HFM). PCFT is also widely expressed in human solid tumors where it contributes to the transport of pemetrexed and other antifolates. This study defines the basis for the functional changes due to a P425R mutation detected in a subject with HFM. Among various substitutions, only positively charged mutants (P425R and P425K) lost function but in a highly selective manner. Transport of reduced folates mediated by P425R-PCFT was virtually abolished; the methotrexate influx Kt was increased fivefold (from 2 to 10 μM). In contrast, the pemetrexed influx Kt mediated by P425R-PCFT was decreased 30% compared with wild-type (WT)-PCFT. Methotrexate inhibition of pemetrexed influx was competitive with a Ki for WT-PCFT comparable to its influx Kt. However, the methotrexate influx Ki for P425R-PCFT was ∼15-fold higher than the WT-PCFT influx Kt and threefold higher than the methotrexate influx Kt for the P425R-PCFT mutant. The confirmed secondary structure and homology modeling place the P425 residue at the junction of the 6th external loop and 12th transmembrane domain, remote from the aqueous translocation pathway, a prediction confirmed by the failure to label P425C-PCFT with N-biotinylaminoethyl methanethiosulfonate-biotin and the absence of inhibition of P425C-PCFT function by water-soluble sulfhydryl reagents. Hence, despite its location, the P425R-PCFT mutation produces a conformational change that fully preserves pemetrexed binding but markedly impairs binding of methotrexate and other folates to the carrier.

scholarly journals Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells ofEscherichia coli

1998 ◽  
Vol 180 (23) ◽  
pp. 6107-6116 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Mirjam E. G. Aarsman ◽  
Arend H. J. Kolk ◽  
Miguel Vicente ◽  
Nanne Nanninga
Keyword(s):  
Cell Division ◽  
Cytoplasmic Membrane ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Domain ◽  
Permissive Temperature ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Division Site ◽  
Periplasmic Domain ◽  
Cell Division Protein

ABSTRACT The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.

scholarly journals Topology and Function of CcmD in Cytochrome c Maturation

2008 ◽  
Vol 190 (10) ◽  
pp. 3489-3493 ◽  
Author(s):  
Cynthia L. Richard-Fogal ◽  
Elaine R. Frawley ◽  
Robert G. Kranz
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Cytochrome C Maturation ◽  
Atp Binding Cassette Transporter ◽  
C Terminus ◽  
N Terminus ◽  
Small Polypeptide ◽  
And Function

ABSTRACT The system I cytochrome c biogenesis pathway requires CcmD, a small polypeptide of 69 residues in Escherichia coli. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain.

scholarly journals Removing the Polyanionic Cargo Requirement for Assembly of Alphavirus Core-Like Particles to Make an Empty Alphavirus Core

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 846
Author(s):  
Julie M. Button ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Nucleic Acid ◽  
Capsid Protein ◽  
Electrostatic Interactions ◽  
Wild Type ◽  
N Terminus ◽  
Charged Residues ◽  
Size And Morphology ◽  
Positively Charged

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form “empty” particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These “empty” particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes.

scholarly journals Positively Charged Termini of the L2 Minor Capsid Protein Are Necessary for Papillomavirus Infection

2001 ◽  
Vol 75 (21) ◽  
pp. 10493-10497 ◽  
Author(s):  
Richard B. S. Roden ◽  
Patricia M. Day ◽  
Brian K. Bronzo ◽  
William H. Yutzy ◽  
Yanqin Yang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Viral Genome ◽  
Bovine Papillomavirus ◽  
Wild Type ◽  
Minor Capsid Protein ◽  
Papillomavirus Infection ◽  
Terminal Amino ◽  
Genome Encapsidation ◽  
Positively Charged

ABSTRACT Coexpression of bovine papillomavirus L1 with L2 mutants lacking either eight N-terminal or nine C-terminal amino acids that encode positively charged domains resulted in wild-type levels of viral genome encapsidation. Despite wild-type binding to the cell surface, the resulting virions were noninfectious. An L2 mutant encoding a scrambled version of the nine C-terminal residues restored infectivity, in contrast to an L2 mutant encoding a scrambled version of the N-terminal residues.

scholarly journals Mechanism of Tripeptide Trimming by γ-Secretase

2021 ◽  
Author(s):  
Apurba Bhattarai ◽  
Sujan Devkota ◽  
Hung Nguyen Do ◽  
Jinan Wang ◽  
Sanjay Bhattarai ◽  
...  
Keyword(s):  
Experimental Data ◽  
Structural Changes ◽  
Transmembrane Domain ◽  
Amyloid Β ◽  
Structural Rearrangements ◽  
Accelerated Molecular Dynamics ◽  
Endoproteolytic Cleavage ◽  
N Terminus ◽  
Conformational Rearrangements ◽  
Positively Charged

The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid β-peptides (Aβ) of Alzheimer's disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (Aβ49 to Aβ46) of wildtype (WT) and familial AD (FAD) mutant APP substrate. In comparison to WT APP, the efficiency of this first trimming step was slightly higher for the I45F, A42T and V46F APP FAD mutants, but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate tripeptide trimming of Aβ49 by γ-secretase. The starting structure was active γ-secretase bound to Aβ49 and APP intracellular domain (AICD), as generated from our previous study that captured activation of γ-secretase for the initial endoproteolytic cleavage of APP (Bhattarai et al., ACS Cent Sci, 2020, 6:969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of Aβ49 to Aβ46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and Aβ49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming by γ-secretase.

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