<scp>NMR</scp> observation of <scp>HIV</scp>‐1 gp120 conformational flexibility resulting from V3 truncation

Moseri, Adi; Schnur, Einat; Noah, Eran; Zherdev, Yuri; Kessler, Naama; Sinha, Eshu Singhal; Abayev, Meital; Naider, Fred; Scherf, Tali; Anglister, Jacob

doi:10.1111/febs.12839

Cited by 8 publications

(24 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Binding of ECL2S to mut gp120 core (gp120 core molecule lacking V1, V2 and V3 containing four mutations that alleviated the aggregation problem) and to mut gp120 core (+V3) ( mut gp120 core lacking only V1 and V2) in the presence or absence of a 27‐residue CD4 peptide mimic, CD4M33 , was measured using surface plasmon resonance (SPR) at 25 °C. Biotin was added to the peptide's N‐terminus to enable immobilization of the peptide on a streptavidin‐coated SPR chip.…”

Section: Resultsmentioning

confidence: 99%

“…In the presence of CD4M33, a K D of 6 AE 4 9 10 À6 M was determined, offering only a rough estimate of the K D . A reliable K D for mut gp120 core (+V3) was not determined due to partial dimerization of the mut gp120 core (+V3)/CD4M33 complex [26]. Similar K D values for ECL2S binding to the mut gp120 core / CD4M33 were measured at pH 6 and pH 6.5 (K D values of 7 AE 3 and 7.5 AE 3 9 10 À6 M, respectively), suggesting that binding of ECL2S to mut gp120 core / CD4M33 is not influenced by pH between 6 and 7.…”

Section: The Ecl2s Peptide Is Helicalmentioning

confidence: 99%

“…Binding of ECL2S peptide to mut gp120 core Binding of ECL2S to mut gp120 core (gp120 core molecule lacking V1, V2 and V3 containing four mutations that alleviated the aggregation problem) and to mut gp120 core (+V3) ( mut gp120 core lacking only V1 and V2) [26] in the presence or absence of a 27-residue 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 R1 S1 Q1 K1 E1 G1 L1 H1 Y1 T1 S1 S1 S1 The averaged chemical shift value of each residue in a helical conformation, d averaged-helix , was calculated as described previously [21]. CD4 peptide mimic, CD4M33 [27], was measured using surface plasmon resonance (SPR) at 25°C.…”

Section: The Ecl2s Peptide Is Helicalmentioning

confidence: 99%

See 2 more Smart Citations

An extended CCR5 ECL2 peptide forms a helix that binds HIV‐1 gp120 through non‐specific hydrophobic interactions

et al. 2015

Self Cite

View full text Add to dashboard Cite

The chemokine receptor CCR5 serves as a co-receptor for the Human Immunodefficiency Virus type-1, HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing the ECL1 and ECL3 were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study we determined that in aqueous buffer the segment Q188-Q194 in an elongated ECL2 peptide (R168 to K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two dimensional Saturation Transfer Difference NMR spectroscopy and dynamic filtering studies revealed the involvement of Y187, F189, W190 and F193 of the helical segment, in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule and therefore, could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: The Ecl2s Peptide Is Helicalmentioning

confidence: 99%

Section: The Ecl2s Peptide Is Helicalmentioning

confidence: 99%

See 1 more Smart Citation

An extended CCR5 ECL2 peptide forms a helix that binds HIV‐1 gp120 through non‐specific hydrophobic interactions

et al. 2015

Self Cite

View full text Add to dashboard Cite

show abstract

“…When NN‐T20‐NITN binding to mut gp120 core (+V3) and mut gp120 core in complex with CD4M33 was compared, we found that V3 improved NN‐T20‐NITN binding by five‐fold. However, it is possible that the stronger mut gp120 core (+V3)/CD4M33 binding to NN‐T20‐NITN is in part the result of the previously observed aggregation of mut gp120 core (+V3)/CD4M33 . Therefore, this contribution of V3 to gp120 binding to NN‐T20‐NITN, in the presence of CD4M33, may or may not be significant.…”

Section: Resultsmentioning

confidence: 99%

“…As noted above, the NN-T20-NITN portion of the modified T20 peptide corresponds to residues 638-673 of gp41 of HIV-1. To map the segment within the NN-T20-NITN peptide that interacts with gp120, we used 1 H-15 N-heteronuclear single quantum coherence (HSQC) measurements of uniformly 15 N labeled (U-15 N) NN-T20-NITN in the presence and absence of gp120. In general, the 1 H-15 N-HSQC spectrum of a 15 N labeled peptide in complex with a much larger protein discriminates residues within the segment that interact with the protein from those peptide residues outside this region.…”

Section: Nn-t20-nitn Binding To R5 Gp120mentioning

confidence: 99%

The C4 region as a target for HIV entry inhibitors – NMR mapping of the interacting segments of T20 and gp120

et al. 2015

Self Cite

View full text Add to dashboard Cite

The peptide T20, which corresponds to a sequence in the C-terminal segment of the HIV-1 trans-membrane glycoprotein gp41, is a strong entry inhibitor of HIV-1. It has been assumed that T20 inhibits HIV-1 infection by binding to the trimer formed by the N-terminal helical region (HR1) of gp41, preventing the formation of a six helix bundle by the N- and C-terminal helical regions of gp41. In addition to binding to gp41, T20 was found to bind to gp120 of X4 viruses and this binding was suggested to be responsible for an alternative mechanism for HIV-1 inhibition by this peptide. In the present study, T20 also was found to bind R5 gp120. Using NMR spectroscopy, the segments of T20 that interact with both gp120 and a gp120/CD4M33 complex were mapped. A peptide corresponding to the fourth constant region of gp120, sC4, was found to partially recapitulate gp120 binding to T20 and the segment of this peptide interacting with T20 was mapped. Our conclusion is that an amphiphilic helix on the T20 C-terminus, binds through mostly hydrophobic interactions, to a non-polar gp120 surface formed primarily by the C4 region. The ten to a thousand-fold difference between the EC50 of T20 against viral fusion and the affinity of T20 to gp120 implies that binding to gp120 is not a major factor in T20 inhibition of HIV-1 fusion. Nevertheless, this hydrophobic gp120 surface could be a target for anti-HIV therapeutics.

show abstract

Conformation state‐specific monobodies regulate the functions of flexible proteins through conformation trapping

Nakamura,

Amesaka,

Hara

et al. 2023

Protein Science

View full text Add to dashboard Cite

Synthetic binding proteins have emerged as modulators of protein functions through protein‐protein interactions (PPIs). Because PPIs are influenced by the structural dynamics of targeted proteins, investigating whether the synthetic‐binders‐based strategy is applicable for proteins with large conformational changes is important. This study demonstrates the applicability of monobodies (fibronectin type‐III domain‐based synthetic binding proteins) in regulating the functions of proteins that undergo tens‐of‐angstroms‐scale conformational changes, using an example of the A55C/C77S/V169C triple mutant (Adktm; a phosphoryl transfer‐catalyzing enzyme with a conformational change between OPEN/CLOSED forms). Phage display successfully developed monobodies that recognize the OPEN form (substrate‐unbound form), but not the CLOSED form of Adktm. Two OPEN form‐specific clones (OP‐2 and OP‐4) inhibited Adktm kinase activity. Epitope mapping with a yeast‐surface display/flow cytometry indicated that OP‐2 binds to the substrate‐entry pathway of Adktm, whereas OP‐4 binding occurs at another site. SEC‐SAXS analysis indicated that OP‐4 binds to the hinge side opposite to the substrate‐binding site of Adktm, retaining the whole OPEN‐form structure of Adktm. Titration of the OP‐4–Adktm complex with Ap5A, a transition‐state analog of Adktm, showed that the conformational shift to the CLOSED form was suppressed although Adktm retained the OPEN‐form (i.e., substrate‐binding ready form). These results show that OP‐4 captures and stabilizes the OPEN‐form state, thereby affecting the hinge motion. These experimental results indicate that monobody‐based modulators can regulate the functions of proteins that show tens‐of‐angstroms‐scale conformational changes, by trapping specific conformational states generated during large conformational change process that is essential for function exertion.This article is protected by copyright. All rights reserved.

show abstract

NMR observation of HIV‐1 gp120 conformational flexibility resulting from V3 truncation

Cited by 8 publications

References 47 publications

An extended CCR5 ECL2 peptide forms a helix that binds HIV‐1 gp120 through non‐specific hydrophobic interactions

An extended CCR5 ECL2 peptide forms a helix that binds HIV‐1 gp120 through non‐specific hydrophobic interactions

The C4 region as a target for HIV entry inhibitors – NMR mapping of the interacting segments of T20 and gp120

Conformation state‐specific monobodies regulate the functions of flexible proteins through conformation trapping

Contact Info

Product

Resources

About