Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

Abstract

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

HIV-1 Gag Non-Cleavage Site PI Resistance Mutations Stabilize Protease/Gag Substrate Complexes In Silico via a Substrate-Clamp

“…Support for the PR/MA-CA polyprotein ternary complex (Figure 1D) comes from small-angle neutron scattering experiments on a Gag polyprotein in which the MA and CA subdomains were found to be closely associated [33], while solution NMR studies of a Gag MA-CA polyprotein revealed that the MA and CA subdomains aligned with complementary surfaces and charges to within 2-6 A in three models [34]. The published experimental results on PR binding to Gag cleavage sites [23][24][25], and Gag MA and CA subdomains associations [33,34] support the structure-based model of HIV-1 PR bound to the MA-CA polyprotein in Figure 1D [23]. The HIV-1 PR S-groove [22], ribbon backbone with subdomains indicated left-to-right (MA yellow 1L6N.pdb, CA bronze 1L6N.pdb and 4XFX.pdb, NC yellow 1MFS.pdb, p6 bronze 2C55.pdb as modified [22]), SP1 and SP2 regions in black, five Gag cleavage sites indicated as are the non-cleavage site residues MA L75, CA H219, and NC R409, which when mutated resulted in PI resistance (carbon grey, nitrogen blue) [30].…”

“…Support for the PR/MA-CA polyprotein ternary complex (Figure 1D) comes from small-angle neutron scattering experiments on a Gag polyprotein in which the MA and CA subdomains were found to be closely associated [33], while solution NMR studies of a Gag MA-CA polyprotein revealed that the MA and CA subdomains aligned with complementary surfaces and charges to within 2-6 A in three models [34]. The published experimental results on PR binding to Gag cleavage sites [23][24][25], and Gag MA and CA subdomains associations [33,34] support the structure-based model of HIV-1 PR bound to the MA-CA polyprotein in Figure 1D [23]. The HIV-1 PR S-groove model was then expanded to include many other retroviral proteases where the native multi-drug resistance was found to be due to a combination of primary active site mutations and secondary S-groove mutations [22].…”

“…The PR S-groove secondary resistance mutations restore PR binding to substrates by increasing S-groove affinity for cleavage site residues P12-P5/P5′-P12′ (|P12-P5|) [22,23], and that in turn restores viral replication while maintaining PI resistance [14][15][16][17][18][19][20][21]. The HIV-1 PR S-groove model is supported by computational chemistry results [22,23], experimental results [23,25], as well as NMR results [24]. The solution NMR results by [22]) bound to 24-mer SP1-NC substrate (backbone ribbon), PR with electrostatic surface potential (blue positive, red negative), cleavage site residues P4-P4 (red) bound to active site, cleavage site residues P12-P5/P5 -P12 (|P12-P5|) bound in S-grooves (bronze); (B) PR bound to 24-mer substrate as in (A), PR as backbone ribbon (A-subunit teal, B-subunit blue), seven S-groove residues that interacted with Gag cleavage site residues |P12-P5|in a NMR study by Deshmukh et al [24] are shown in addition to D29 (carbon grey, oxygen red, nitrogen blue), SP1-NC cleavage site residues P5 N and P4 G including backbone nitrogens indicated, active site tetrahedrally coordinated water under flaps; (C) PR bound to 24-mer SP1-NC substrate as in (B), with ten native PR S-groove residues shown, Wensing and coworkers reported PI resistance mutations for each of those ten native residues [13], S-groove residues on PR A-subunit anti-parallel β-sheet (left), residues on backside of PR B-subunit S-groove anti-parallel β-sheet (right), red residue labels indicate primary PI resistance, black residue labels indicate secondary PI resistance, when residues were mutated as reported by Wensing and coworkers [13]; (D) PR bound to MA/CA cleavage site when part of a MA-CA polyprotein (1L6N.pdb as modified [23]) with backbone ribbon (MA yellow, CA bronze) as previously reported [23].…”

“…The PR S-groove secondary resistance mutations restore PR binding to substrates by increasing S-groove affinity for cleavage site residues P12-P5/P5 -P12 (|P12-P5|) [22,23], and that in turn restores viral replication while maintaining PI resistance [14][15][16][17][18][19][20][21]. The HIV-1 PR S-groove model is supported by computational chemistry results [22,23], experimental results [23,25], as well as NMR results [24]. The solution NMR results by Deshmukh and coworkers demonstrated that HIV-1 PR S-groove residues interacted directly with Gag cleavage site residues outside of P4-P4 , i.e., |P12-P5|, Figure 1B, [24].…”

“…In that study by Deshmukh and coworkers, the cleavage rate for the mutated MA/CA site did not change significantly [46], indicating the importance of cleavage site residues P12-P6/P6 -P12 in determining the MA/CA cleavage rate, since both the MA/CA and SP1/NC sites were bordered by structured subdomains that can form a substrate-clamp, see Figures 3, 5 and 6 [46]. Interestingly, Miczi and coworkers demonstrated the importance of the MA/CA cleavage site P12-P6/P5 -P12 residues by replacing the native MA/CA site residues P12-P6/P5 -P12 with the respective residues from the slow CA/SP1 site (also named CA/p2), and reported that the cleavage at the mutant MA/CA site had a 2.9-fold lower k cat /K M [25]. The CA/SP1 cleavage site residues |P12-P5| were reported to have the weakest in silico interaction with PR of all the cleavage site |P12-P5| residues in the Gag and Pol polyproteins [22].…”