Characterization of an A3G-VifHIV-1-CRL5-CBFβ Structure Using a Cross-linking Mass Spectrometry Pipeline for Integrative Modeling of Host–Pathogen Complexes

Abstract

Structural analysis of host–pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue–protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFβ) to determine the structure of the (A3G-Vif-CRL5-CBFβ) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFβ complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host–pathogen protein complexes.

Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase

“…The structural model of the A3G-Vif complex has been long sought-after since the identification of A3G as a host factor that restricts HIV infection ( 17 ). The A3G-Vif interaction has been expected to be purely protein based, as the binary complex structure has been predicted by biochemistry-aided or cross-linking mass spectrometry–aided computational modeling ( 29 , 42 ). The direct complex formation has been shown for APOBEC3F (A3F) and Vif with the support of a polypeptide linker between A3F and CBF-β ( 43 ).…”

Recent advances in poor HIV immune reconstitution: what will the future look like?

“…Tetherin proteins that mediate MVs transport and contact with receptor membranes can keep microvesicles arrested on the cell surface prevent the release and transmission of HIV-1-carrying extracellular vesicles and, to a certain extent, alleviate the associated inflammatory disease ( Weber et al, 2020 ). A3G is a human cytidine deaminase that can potentially hypermutate viral genomes, causing impairment of reverse transcriptase activity during the retrotranscription process ( Kaake et al, 2021 ; Chesarino and Emerman, 2022 ). Tetherin and A3G expressions might be increased by EVs at the mRNA level of cellular restriction factors ( Navarrete-Muñoz et al, 2021 ).…”

Integrative structure determination of histones H3 and H4 using genetic interactions

“…We have achieved progress in developing methods for integrative structure determination of host-pathogen complexes, primarily based on cross-linking data [82][83][84]. However, structural characterization of host-pathogen protein assemblies remains challenging, largely due to their compositional and conformational heterogeneity.…”