Binding kinetics and substrate selectivity in HIV-1 protease-Gag interactions probed at atomic resolution by chemical exchange NMR

Proc Natl Acad Sci U S A. 2017 Nov 14;114(46):E9855-E9862. doi: 10.1073/pnas.1716098114. Epub 2017 Oct 30.


The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr-Purcell-Meiboom-Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease-Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.

Keywords: chemical exchange saturation transfer; conformational dynamics; invisible states; relaxation dispersion; substrate selection.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acid Sequence
  • Biophysical Phenomena
  • Carrier Proteins / chemistry
  • Carrier Proteins / pharmacokinetics*
  • Catalytic Domain
  • Drug Resistance, Viral / genetics
  • HIV Protease / chemistry*
  • HIV Protease / genetics
  • HIV Protease / pharmacokinetics*
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Kinetics
  • Magnetic Resonance Imaging
  • Magnetic Resonance Spectroscopy / methods*
  • Models, Molecular
  • Mutagenesis
  • Protein Binding
  • Protein Conformation
  • Protein Domains
  • Protein Interaction Domains and Motifs*
  • Proteolysis
  • Recombinant Proteins
  • Substrate Specificity
  • gag Gene Products, Human Immunodeficiency Virus / chemistry*
  • gag Gene Products, Human Immunodeficiency Virus / genetics
  • gag Gene Products, Human Immunodeficiency Virus / pharmacokinetics*


  • Carrier Proteins
  • Recombinant Proteins
  • gag Gene Products, Human Immunodeficiency Virus
  • HIV Protease
  • p16 protease, Human immunodeficiency virus 1