Molecular Dynamics Simulation Reveals Exposed Residues in the Ligand-Binding Domain of the Low-Density Lipoprotein Receptor that Interacts with Vesicular Stomatitis Virus-G Envelope

Viruses. 2019 Nov 15;11(11):1063. doi: 10.3390/v11111063.

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disease most often caused by mutations in the low-density lipoprotein receptor (LDLR) gene, which consists of 18 exons spanning 45 kb and codes for a precursor protein of 860 amino acids. Mutations in the LDLR gene lead to a reduced hepatic clearance of LDL as well as a high risk of coronary artery disease (CAD) and sudden cardiac death (SCD). Recently, LDLR transgenes have generated interest as potential therapeutic agents. However, LDLR packaging using a lentiviral vector (LVV) system pseudotyped with a vesicular stomatitis virus (VSV)-G envelope is not efficient. In this study, we modified the LVV system to improve transduction efficiency and investigated the LDLR regions responsible for transduction inhibition. Transduction efficiency of 293T cells with a 5'-LDLReGFP-3' fusion construct was only 1.55% compared to 42.32% for the eGFP construct. Moreover, co-expression of LDLR affected eGFP packaging. To determine the specific region of the LDLR protein responsible for packaging inhibition, we designed constructs with mutations or sequential deletions at the 3' and 5' ends of LDLR cDNA. All constructs except one without the ligand-binding domain (LBD) (pWoLBD-eGFP) resulted in low transduction efficiency, despite successful packaging of viral RNA in the VSV envelope, as confirmed through RT-PCR. When we evaluated a direct interaction between LDLR and the VSV envelope glycoprotein using MD simulation and protein-protein interactions, we uncovered Val119, Thr120, Thr67, and Thr118 as exposed residues in the LDLR receptor that interact with the VSV protein. Together, our results suggest that the LBD of LDLR interacts with the VSV-G protein during viral packaging, which significantly reduces transduction efficiency.

Keywords: CHARMM; Gromacs; I-TASSER; MOPAC2009; Molecular Operating Environment; coronary artery disease; familial hypercholesterolemia; fusion protein; lentiviral vector system; low-density lipoprotein receptor (LDLR); molecular dynamics simulation; pyDock; sudden cardiac death; transduction; transfection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Line
  • Genes, Reporter
  • Humans
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / metabolism
  • Molecular Docking Simulation*
  • Molecular Dynamics Simulation*
  • Mutation
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs*
  • Receptors, LDL / chemistry*
  • Receptors, LDL / metabolism
  • Recombinant Fusion Proteins
  • Viral Envelope Proteins / chemistry*
  • Viral Envelope Proteins / metabolism

Substances

  • G protein, vesicular stomatitis virus
  • Membrane Glycoproteins
  • Receptors, LDL
  • Recombinant Fusion Proteins
  • Viral Envelope Proteins