Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

Abstract

The assembly of two monomeric constructs spanning segments 1-199 (MPro^1-199) and 10-306 (MPro^10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro^1-199 and MPro^10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro^10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro^1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro^10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 μM MPro^1-199. Catalytic activity arises primarily from MPro^1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro^1-199 and E290 of MPro^10-306. Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

Keywords: SARS-CoV-2 main protease; alternate folding pathways; dimer interface; fold-switching; inhibitor binding; monomer-dimer equilibrium; precursor processing; protein folding; protein fragment assembly; structure comparison; swapped dimer.

Figure 1

Domain organization of mature MPro dimer and rationale for this investigation.A, one monomer [PDB 7JUN, (15)] is shown in white. In the other, the catalytic region composed of domains I and II and the helical region (domain III) are colored goldenrod and yellow green, respectively. The connecting loop between the catalytic and helical regions and the N-terminal residues 1 to 9 (N-finger) are shown in black. The catalytic dyad H41/C145 residues (red) are shown as ball and stick representations and the active site oxyanion loop in red. B, steps in the mechanism of MPro maturation from its precursor analog (4). C, the two deletion constructs used in this study to examine the transient pathway for MPro subunit assembly concomitant with the appearance of enhanced catalytic activity (4). Residues 1 to 199 make up the catalytic domain represented by the construct MPro^1-199. Ovals, MPro catalytic and helical domains; black line, MPro N-terminal residues; dashed black and continuous blue lines, flanking nsp4 and nsp5 regions, respectively; red circles, catalytic dyad residues; E∗, active (wound) conformation of the active site oxyanion loop.

Figure 2

Assembly of MPro^1-199and MPro^10-306monitored by SV-AUC. Absorbance c(s) distribution in the absence (A) and presence (B–D) of the inhibitor in buffer B at 25 °C. Proteins and inhibitor were prepared at three concentrations (12.5–50 μM) as shown in B through D. Actual measured concentrations by AUC are listed beside each plot. Estimated S and corresponding mass values are listed in Table 1.

Figure 3

Molecular mass estimation of MPro^1-199and MPro^10-306association by SEC-MALS.A, Fifty micromolars of each protein was mixed either in the absence (blue trace) or presence of 10-fold molar excess of NMV (black) in a total injection volume of 125 μl. Black (without NMV) and red (with NMV) circles indicate estimated mass range. B, Hundred micromolars of each protein mixed with 10-fold molar excess of NMV (black) in a total injection volume of 140 μl. Samples were fractionated on Superose-12 column (1 × 30 cm) in buffer B at a flow rate of 0.5 ml/min at 25 °C. Red circles indicate estimated mass range. C, collected fractions from (B, 10.5–11.5 ml) showing complex formation were analyzed by SDS-PAGE. The estimated ratio (2:1.4) by quantifying the band intensities indicates a complex approaching a stoichiometry of two molecules of MPro^10-306 and one molecule of MPro^1-199 at ∼2.5 μM concentration estimated at the top of the peak. This ratio was confirmed by the analyses of predetermined mixed amounts (2:2, 2:1 and 1:2) of the two proteins (where 2 denotes 1 μg of protein) by SDS-PAGE. M and kDa denote molecular weight standards and kilo Daltons, respectively. Molecular mass was calculated with the Astra software provided with the instrument.

Figure 4

Catalytic activity of various assembled compositions of MPro^1-199and MPro^10-306and their mutants. The concentrations of each protein mixed at a ratio as indicated above the plots are (A, E) 25 μM and (B) 10 μM. A, bar plot of the initial rates calculated from progress curves of catalytic activity in the absence and presence of GC373, respectively, shown in Fig. S2. B, a plot of the rate versus GC373 concentration showing the rise and fall in catalytic activity of the protein mixture. C, plots of the catalytic activity of 40 μM MPro^1-199 mixed with 80 μM GC373 versus increasing concentration of MPro^10-306 (red) and vice versa (black). D, a plot of the rate/MPro^1-199 concentration versus MPro^1-199 concentration showing the mid-point of the dissociation of the complex (∼6 μM, dashed intercept) accompanied by the loss in catalytic activity. E, SV-AUC analysis of similar mixtures with GC373 as shown in (A). kDa denotes kilo Daltons.

Figure 5

SEC profiles of atypical dimer forms of MPro^10-306and MPro^197-306, mass estimations, and conversion to the monomer.A and C, expressed proteins after initial purification by NAC (10–12 mg) were subjected to SEC on Superose-12 column in buffer B at ambient temperature. B and D, SEC-MALS analyses of atypical dimer and monomer forms. Estimated concentrations at the top of the peak are indicated. Injection concentrations for SEC-MALS range from 25 to 336 μg in 125 μl. Conversion of the MPro^10-306 atypical dimer to the monomer [traces other than red in (B)] was monitored by injecting 1/10th the injection concentration of 250 μg in 125 μl (red) following incubation at 25 and 37 °C for the specified duration. Tbin and kDa denote thrombin and kilo Daltons, respectively.

Figure 6

SEC profiles of MPro^WTand MPro^H41Aexpressed as precursors appended to N-terminal nsp4 residues.A and B, expressed proteins after initial purification by NAC [(A): 6 mg, (B): 2 mg)] were subjected to SEC on Superose-12 column in buffer B at ambient temperature. ^(-102) and ^(-6) denote the length of nsp4 residues and asterisk, the Q to E mutation (see Table S1 for details). C and D, pooled peak fractions of dimer and monomer from (A) and (B) were subjected to SEC-MALS as described in Experimental procedures. Estimated concentrations at the top of the peak are indicated. Injection concentrations for SEC-MALS were 250 μg in 125 μl. Conversion of the ^(-6∗)MPro^H41A atypical dimer to the monomer [traces other than red in (C)] was monitored by injecting 1/10th the injection concentration of 250 μg in 125 μl (red) following incubation at 25 °C for the specified duration. kDa denotes kilo Daltons.

Figure 7

Preserved interface contacts upon association of MPro^1-199and MPro^10-306exhibiting possible alternate conformation of the helical domain.A and B, previously described alternate [via fold-switching (FS)] conformation of isolated SARS-CoV helical domain [B, PDB: 3EBN, (28)] compared with the helical domain of SARS-CoV-2 MPro^WT native dimer [A, PDB: 7JUN, (15)]. The five helices are labeled α₁ to α₅. C, overlay of MPro^C145A dimer [white, PDB: 7N89, (35)] and FS-dimer of the helical domain [blue and yellow, PDB: 3EBN, (28)]. The box highlights the region critical for dimer interface stability of mature MPro. D, despite the helical residues 201 to 214 switching (α₁) their location to the opposite subunit, the positioning of the helix (293–301, α₅) with critical inter-monomer E290/R4’ (prime denotes the second subunit) is clearly maintained as in the native conformation, which does not exhibit fold-switching.

Figure 8

Predicted structures formed upon mixing MPro^1-199and MPro^10-306.A, room temperature X-ray structure of MPro^WT [gold, PDB: 7JUN, (15)] overlayed on cryo-X-ray monomer structure of SARS-CoV MPro^R298A [white, PDB: 2QCY, (16)]. The inactive conformation of the active site oxyanion loop and the rotated helical domain (residues 200–306), relative to the WT dimer, are shown in orange red. B and C, expected folded conformation of purified MPro^10-306 and MPro^1-199 prior to mixing either in the absence or presence of inhibitor. The scheme for their association is shown. FS denotes fold-switch. D and E, progressive assembly upon increasing protein concentration showing a stoichiometry of 1:1 (D), 2:1 (E), and 2:2 (F) of MPro^10-306:MPro^1-199. Theoretical molecular weights of the complexes are indicated in kilo Daltons (kDa). Molecular structure models were created using SARS-CoV MPro^WT octamer [PDB: 3IWM, (23)], helical domain of SARS-CoV [PDB: 3EBN, (28)], and SARS-CoV-2 MPro^WT and MPro^C145A [PDB: 7JUN and 7N89, respectively, (15, 35)].

Figure 9

Predicted structures formed upon mixing MPro^1-199and MPro^10-306. AlphaFold 3 model for the 2:1 complex of MPro^10-306 and MPro^1-199 showing the association of domain III through intermonomer contacts formed by residues 280 and 283 to 286 and the E290/R4′ salt bridge like that of the WT mature homodimer. All AlphaFold 3 generated assemblies are shown in Fig. S3.