A Unified Mechanism for Aminopeptidase N-based Tumor Cell Motility and Tumor-homing Therapy

Liu, Chang; Yang, Yang; Chen, Lang; Lin, Ying Wu; Li, Fang

doi:10.1074/jbc.m114.566802

Cited by 36 publications

(40 citation statements)

References 41 publications

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“…Protein-Protein Binding Assay Using AlphaScreen-The interactions between recombinant MHV S1-NTD and recombinant mCEACAM1a or mCEACAM1b (wild type or mutant) were measured using AlphaScreen as previously described (46,47). Briefly, 300 nM MHV S1-NTD with a C-terminal His 6 tag was mixed with 30 nM mCEAMCAM1a or mCEACAM1b (wild type or mutant) with a C-terminal human IgG 4 Fc tag in Opti-Plate-96 (PerkinElmer Life Sciences) for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor

Peng

Yang

Pasquarella

et al. 2017

Journal of Biological Chemistry

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Hosts and pathogens are locked in an evolutionary arms race. To infect mice, mouse hepatitis coronavirus (MHV) has evolved to recognize mouse CEACAM1a (mCEACAM1a) as its receptor. To elude MHV infections, mice may have evolved a variant allele from the gene, called, producing mCEACAM1b, which is a much poorer MHV receptor than mCEACAM1a. Previous studies showed that sequence differences between mCEACAM1a and mCEACAM1b in a critical MHV-binding CC' loop partially account for the low receptor activity of mCEACAM1b, but detailed structural and molecular mechanisms for the differential MHV receptor activities of mCEACAM1a and mCEACAM1b remained elusive. Here we have determined the crystal structure of mCEACAM1b and identified the structural differences and additional residue differences between mCEACAM1a and mCEACAM1b that affect MHV binding and entry. These differences include conformational alterations of the CC' loop as well as residue variations in other MHV-binding regions, including β-strands C' and C'' and loop C'C''. Using pseudovirus entry and protein-protein binding assays, we show that substituting the structural and residue features from mCEACAM1b into mCEACAM1a reduced the viral receptor activity of mCEACAM1a, whereas substituting the reverse changes from mCEACAM1a into mCEACAM1b increased the viral receptor activity of mCEACAM1b. These results elucidate the detailed molecular mechanism for how mice may have kept pace in the evolutionary arms race with MHV by undergoing structural and residue changes in the MHV receptor, providing insight into this possible example of pathogen-driven evolution of a host receptor protein.

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Section: Methodsmentioning

confidence: 99%

Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor

Peng

Yang

Pasquarella

et al. 2017

Journal of Biological Chemistry

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“…The quality of the final model was analyzed with Mol-Probity [43] and EMRinger [44]. The validation statistics of the structural models are summarized in S1 Table. AlphaScreen protein-protein binding assay AlphaScreen protein-protein binding assay was carried out between recombinant MHV S1-NTD and recombinant CEACAM1a and between recombinant MHV S-e and recombinant CEACAM1a as described previously [45,46]. Briefly, Fc-tagged CEACAM1a (at 6 nM final concentration) was incubated with either His 6 -tagged MHV S1-NTD or His 6 -tagged MHV S-e (at 100 nM final concentration) in ½ AreaPlate (PerkinElmer, Waltham, MA) at room temperature for 1 hour.…”

Section: Model Building and Refinementmentioning

confidence: 99%

Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry

et al. 2020

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Coronaviruses recognize a variety of receptors using different domains of their envelopeanchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to postfusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses. Author summaryCoronaviruses recognize many receptors using their envelope-anchored spike protein.The role of receptor binding in coronavirus entry into host cells is a fundamental question in virology. Mouse hepatitis coronavirus (MHV) is unique among all coronaviruses in that it uses the N-terminal domain (NTD) of its spike protein to bind a protein receptor CEACAM1a. While extensive research has been performed on the cell entry mechanisms of coronaviruses that use a different domain of their spike protein for receptor binding, the cell entry mechanism for MHV is still elusive. Here we determined the cryo-EM PLOS PATHOGENS PLOS Pathogens | https://doi., et al. (2020) Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry. PLoS Pathog 16 (3): e1008392. https://doi.org/10.Data Availability Statement: The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMD) under accession code EMD-21377. The atomic model has been deposited in the Protein Data Bank (PDB) under accession code 6VSJ.Funding: This work was supported by R01AI089728 (to F. Li) from National Institute of Allergy and Infectious Diseases (https://www.niaid. nih.gov/). The funders had no role in study design, structure of MHV spike protein complexed with CEACAM1a. The structure reveals unique features of receptor binding by MHV spike that facilitate the structural changes of MHV spike and promote cell entry of MHV. We further confirmed the structural results with biochemical and negative-stain EM analyses. These results suggest that receptor binding plays dual roles in MHV entry: it promotes both viral attachm...

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“…Recently, peptides containing the asparagine-glycine-arginine (Asn-Gly-Arg, NGR) motif [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75] and their deamidation products containing the iso DGR motif [38,41,46,48,56,59,74,76,77,78,79,80,81...…”

Section: Introductionmentioning

confidence: 99%

Succinimide Formation from an NGR-Containing Cyclic Peptide: Computational Evidence for Catalytic Roles of Phosphate Buffer and the Arginine Side Chain

Kirikoshi

Manabe

Takahashi

2017

IJMS

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The Asn-Gly-Arg (NGR) motif and its deamidation product isoAsp-Gly-Arg (isoDGR) have recently attracted considerable attention as tumor-targeting ligands. Because an NGR-containing peptide and the corresponding isoDGR-containing peptide target different receptors, the spontaneous NGR deamidation can be used in dual targeting strategies. It is well known that the Asn deamidation proceeds via a succinimide derivative. In the present study, we computationally investigated the mechanism of succinimide formation from a cyclic peptide, c[CH2CO-NGRC]-NH2, which has recently been shown to undergo rapid deamidation in a phosphate buffer. An H2PO4− ion was explicitly included in the calculations. We employed the density functional theory using the B3LYP functional. While geometry optimizations were performed in the gas phase, hydration Gibbs energies were calculated by the SM8 (solvation model 8) continuum model. We have found a pathway leading to the five-membered ring tetrahedral intermediate in which both the H2PO4− ion and the Arg side chain act as catalyst. This intermediate, once protonated at the NH2 group on the five-membered ring, was shown to easily undergo NH3 elimination leading to the succinimide formation. This study is the first to propose a possible catalytic role for the Arg side chain in the NGR deamidation.

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A Unified Mechanism for Aminopeptidase N-based Tumor Cell Motility and Tumor-homing Therapy

Cited by 36 publications

References 41 publications

Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor

Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor

Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry

Succinimide Formation from an NGR-Containing Cyclic Peptide: Computational Evidence for Catalytic Roles of Phosphate Buffer and the Arginine Side Chain

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