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

Mol Cell Proteomics. 2021;20:100132. doi: 10.1016/j.mcpro.2021.100132. Epub 2021 Aug 11.

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.

Keywords: architectures of host–pathogen complexes; cross-linking mass spectrometry; integrative structure modeling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • APOBEC-3G Deaminase / chemistry*
  • Core Binding Factor beta Subunit / chemistry*
  • Cullin Proteins / chemistry*
  • Mass Spectrometry
  • Models, Molecular
  • Ubiquitin-Protein Ligases / chemistry*
  • vif Gene Products, Human Immunodeficiency Virus / chemistry*

Substances

  • Core Binding Factor beta Subunit
  • Cullin Proteins
  • vif Gene Products, Human Immunodeficiency Virus
  • vif protein, Human immunodeficiency virus 1
  • Ubiquitin-Protein Ligases
  • APOBEC-3G Deaminase