Insights into the oligomeric structure of the HIV-1 Vpu protein

Majeed, Saman; Adetuyi, Oluwatosin; Borbat, Peter P.; Islam, Ataul; Ishola, Olamide; Zhao, Bo; Georgieva, Elka R.

doi:10.1016/j.jsb.2023.107943

Cited by 10 publications

(35 citation statements)

References 71 publications

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“…Of these, the SEC peak of F1 was well-defined and corresponds to protein of molecular weight greater than 200 kDa and F2 was a combination of two peaks containing proteins of molecular weight greater than 100-150 kDa. 28 Both F1 and F2 were WB-positive, thus containing apoAI-EfpA. This result provides one more evidence of apoAI-EfpA’s homo-oligomerization in detergent, but these oligomers a somewhat heterogeneous ranging from tetramers to lower order oligomers.…”

Section: Resultsmentioning

confidence: 64%

“…It is worth noting that the F1 peak occurs well after the void volume of 6 ml for the column we used for SEC. 28 Similarly to Ni+Co-affinty purified protein, the SDS-PAGE and WB of the F1 and F2 (Figure 2C), showed protein bands at molecular weight of monomeric and oligomeric FL AI-EfpA. However, WB-positive protein bands corresponding to molecular weight lower than that of the FL apoAI-EfpA ware also observed.…”

Section: The Expressed In E Coli Chimera Construct Apoai-efpa Is a Me...mentioning

confidence: 78%

“…These GNPs bind to the protein His-tag. 28 The protein-to-GNPs molar ratios were ca. 1:130 and 1:100 for protein in detergent and in lipid, respectively.…”

Section: Preparation Of Samples For Nsem and Nsem Imaging Of The Apoa...mentioning

confidence: 99%

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Protein engineering, production, reconstitution in lipid nanoparticles, and initial characterization of theMycobacterium tuberculosisEfpA drug exporter

Ishola,

Ogunbowale,

Islam

et al. 2023

Preprint

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Mycobacterium tuberculosis (Mtb) drug exporters contribute an efficient mechanism for drug resistance. Therefore, understanding the structure/function relationship in these proteins is important. We focused on the Mtb EfpA efflux pump, which belongs to the major facilitator superfamily (MSF) and transports anti-tuberculosis drugs outside the bacterial cell. Here, we report on our advancements in producing and in vitro characterization of this protein. We engineered a construct of apolipoprotein AI (apoAI) fused to the N-terminus of EfpA (apoAI-EfpA) and cloned it in an E. coli expression vector. This fusion construct was found in a membrane-bound form, unlike the deposited in inclusion bodies EfpA without apoAI. We purified the apoAI-EfpA in detergent to a sufficient degree and reconstituted it in DOPC/DOPS lipids. We found that upon reconstitution in lipid, the apoAI-EfpA forms discoidal protein-lipid nanostructures with a diameter of about 20 nm, resembling nanodiscs. We further detected apoAI-EfpA form oligomers in β-DDM and lipid. To the best of our knowledge, this is the first complete protocol on the expression, purification, and lipid reconstitution of the Mtb EfpA reported. Our bioinformatic analysis confirmed the earlier proposed 14-transmembrane helices of the Mtb EfpA. We also found very high identity, >80%, among the EfpAss of diverse mycobacterial species. Outside of mycobacteria, EfpA has no close homologues with only low identity with the QacA family of transporters. These findings possibly indicate high specificity of EfpA mechanisms. Our developments provide a foundation for more comprehensive in vitro studies on the EfpA exporter.

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Section: Resultsmentioning

confidence: 64%

Section: The Expressed In E Coli Chimera Construct Apoai-efpa Is a Me...mentioning

confidence: 78%

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Protein engineering, production, reconstitution in lipid nanoparticles, and initial characterization of theMycobacterium tuberculosisEfpA drug exporter

Ishola,

Ogunbowale,

Islam

et al. 2023

Preprint

Self Cite

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“…Within the literature on Vpu, there is still ongoing debate on its oligomeric state. ,− There is currently no structural information on the full length Vpu forming an oligomer, but there has been one structure solved of full-length Vpu in its monomeric form. ,, Some previous studies have suggested that Vpu assembles into fixed pentameric complexes, whereas other studies found that Vpu assembles into a broader range of oligomers. ,− Interestingly, some previous studies suggested that higher solution ionic strengths drive Vpu to assemble into large oligomeric complexes …”

Section: Resultsmentioning

confidence: 99%

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

Townsend,

Fapohunda,

Wang

et al. 2024

Biochemistry

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Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can selfassemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.

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“…Within the literature on Vpu, there is still ongoing debate on its oligomeric state. 31,[49][50][51][52] There is currently no structural information of the full length Vpu forming an oligomer, but there has been one structure solved of full-length Vpu in its monomeric form. 37,53,54 Some previous studies have suggested that Vpu assembles into fixed pentameric complexes, 55 whereas other studies found that Vpu assembles into a broader range of oligomers.…”

Section: Hiv-vpumentioning

confidence: 99%

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

Townsend,

Fapohunda,

Wang

et al. 2023

Preprint

View full text Add to dashboard Cite

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry (MS) in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for mechanisms of their biological functions as well as their potential as therapeutic targets.

show abstract

Insights into the oligomeric structure of the HIV-1 Vpu protein

Cited by 10 publications

References 71 publications

Protein engineering, production, reconstitution in lipid nanoparticles, and initial characterization of theMycobacterium tuberculosisEfpA drug exporter

Protein engineering, production, reconstitution in lipid nanoparticles, and initial characterization of theMycobacterium tuberculosisEfpA drug exporter

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

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