Model

Overview

The models have played a crucial role in guiding and assisting our project this year. Our models can be divided into three parts: the first part is the structural prediction of GST-nsp5, the second part is the enzymatic kinetics of nsp5, and the third part is the rational design of nsp5. Through the establishment and analysis of these models, we have achieved the following goals:

1. Determined the reason why HRV 3C protease could not cleave the N-terminal GST tag of nsp5 and how the GST tag might affect nsp5 enzymatic activity.
2. Characterized the enzymatic kinetics of nsp5, determining its k_cat/K_M to be 27,691 s⁻¹M⁻¹.
3. Based on structural prediction, we rationally designed a nsp5 mutant, nsp5-T21I, which enhances substrate interaction and increases enzymatic activity, and finally determined its k_cat/K_M to be 35,069 s⁻¹M⁻¹.

Structure prediction of GST-nsp5

We initially used the vector pGEX-GST-nsp5-His to express nsp5, but during the purification process, we found that the N-terminal GST tag could not be cleaved by HRV 3C protease. To investigate the cause of this phenomenon, we used AlphaFold3 to predict the structure of GST-nsp5 (Figure 1)

Since nsp5 functions as a dimer^[1], we input two copies of GST-nsp5 for structure prediction. The results revealed that GST-nsp5 forms a centrosymmetric dimer, and upon closely examining the HRV 3C protease recognition region (shown in cyan), we observed that this sequence folds inward into the nsp5 structure, with the cleavage site residues 226Q and 227G positioned at the deepest part of the nsp5 cavity. This suggests that nsp5 directly blocks HRV 3C protease from accessing the cleavage site, thereby preventing the removal of the N-terminal GST tag.

We also investigated whether the presence of the GST tag might affect nsp5 enzymatic activity. To do so, we compared the structures of GST-nsp5 and the nsp5-substrate complex^[2] (PDB: 7DVP) (Figure 2). Interestingly, we found that the HRV 3C protease recognition region (shown in cyan) overlaps with the site where nsp5 recognizes its natural substrate (shown in violet). This indicates that the presence of the GST tag and the HRV 3C protease recognition sequence may interfere with substrate recognition by nsp5. This prompted us to further design a vector, pGEX-GST-nsp5_native-His, to express nsp5 with a native N- and C-terminus (see Engineering Success for details).

Enzymatic kinetics of nsp5

After obtaining the purified nsp5, we aimed to thoroughly characterize it as a new iGEM part. Nsp5 plays a crucial role in the SARS-CoV-2 life cycle by recognizing and cleaving multiple sites on the viral polyprotein, thereby releasing other non-structural proteins^[3] (Figure 3). This process can be described by the classic Michaelis-Menten equation.

The Michaelis-Menten equation describes the relationship between the initial velocity of an enzyme-catalyzed reaction and the substrate concentration:

In this equation, V0 represents the current reaction rate, Vmax represents the maximum reaction rate, [E] denotes the total enzyme concentration, indicates the current substrate concentration, K_m is the Michaelis constant that reflects the affinity between the enzyme and the substrate, and k_cat represents the amount of substrate catalyzed by the enzyme per unit time. Typically, k_cat/K_m is used to describe the efficiency of enzyme-catalyzed reactions.

To determine the K_m and k_cat of nsp5, we designed a FRET-based fluorescent probe (see Design). First, we fixed the nsp5 concentration (1.2 μM) and set a gradient of substrate concentrations (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) for the reaction. After the reaction concluded, we fitted the fluorescent intensity of the reaction over the initial 40 seconds using a linear equation (Figure 4), where the slope represents the change in fluorescent intensity over time and can be considered the reaction velocity V₀ (Table 1).

Finally, we applied the Michaelis-Menten equation to fit the reaction velocities at different substrate concentrations, resulting in the determination of K_m and k_cat values for nsp5 (Figure 5).

In summary, we derived the kinetic equation for nsp5:

The catalytic efficiency of nsp5 was determined to be k_cat/K_m = 27,691 s^-1M^-1 .

Rational design of nsp5

Since nsp5 has the potential to be developed into a tool enzyme, we aimed to enhance its enzymatic activity through rational design. Our strategy was to introduce mutations in nsp5 that could strengthen its binding affinity to the linker substrate (N-GSAVLQSGFRK-C), thereby improving nsp5's activity. Given that the catalytic center of the enzyme is relatively conserved, mutations in the catalytic core often lead to loss of function. Therefore, we chose to modify amino acids that are relatively distant from the catalytic center but still involved in substrate binding. Additionally, to facilitate comparisons of interactions before and after mutation, we focused on amino acids with relatively simple interactions with the substrate.

First, we predicted the structure of the wild-type nsp5 in complex with the linker substrate. Through structural analysis, we found that T21_nsp5 is distant from the catalytic center and interacts with only one amino acid of the substrate. Therefore, we chose to modify this site.

To enhance the interaction between T21_nsp5 and the substrate, we aimed to replace T21_nsp5 with an amino acid that has a more extended side chain, while retaining the original characteristics of the side chain. For this purpose, we chose to mutate T to I. This mutation replaces the hydroxyl group attached to the carbon atom of the R-group with a -CH₂-CH₃ group, increasing the side chain's length.

We then predicted the structure of the nsp5-T21I mutant in complex with the same linker substrate and performed a comparative analysis with the wild-type nsp5. The results showed that the overall structures of the two were very similar (Cα RMSD = 0.16), with only the R10 residue of the substrate(R10_substrate) exhibiting a rotation of approximately 50 degrees. Therefore, we focused on analyzing this region in detail (Figure 6).

By comparing the structures, we found that when T21_nsp5 was replaced by I21_nsp5, the R10_substrate side chain, which previously interacted with T21_nsp5, was displaced from its original position. Although the interaction with residue 21 was weakened, this subtle conformational change allowed the carbonyl oxygen of G23_nsp5 to form additional interactions with the R10_substrate side chain. Additionally, the distance between the carbonyl oxygens of T24_nsp5 and R10_substrate decreased from 2.8 Å to 2.7 Å.

In summary, in wild-type nsp5, only T24_nsp5 and T21_nsp5 interact with R10_substrate, whereas in nsp5-T21I, residues I21_nsp5, G23_nsp5, and T24_nsp5 together stabilize R10_substrate. We hypothesize that this change enhances the interaction between the substrate and nsp5, leading to improved enzymatic activity.

To further validate our hypothesis, we expressed and purified nsp5-T21I and determined its and based on the previous enzymatic kinetics model. The results showed that the k_cat/K_m of nsp5-T21I was 35,069 s⁻¹M⁻¹, which is higher than that of wild-type nsp5 (27,691 s⁻¹M⁻¹). This confirms that our rationally designed nsp5-T21I indeed possesses higher enzymatic activity compared to wild-type nsp5.

References

1. Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Peng C, Duan Y, Yu J, Wang L, Yang K, Liu F, Jiang R, Yang X, You T, Liu X, Yang X, Bai F, Liu H, Liu X, Guddat LW, Xu W, Xiao G, Qin C, Shi Z, Jiang H, Rao Z, Yang H. Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors. Nature. 2020 Jun;582(7811):289-293. doi: 10.1038/s41586-020-2223-y. Epub 2020 Apr 9. PMID: 32272481.
2. Zhao Y, Zhu Y, Liu X, Jin Z, Duan Y, Zhang Q, Wu C, Feng L, Du X, Zhao J, Shao M, Zhang B, Yang X, Wu L, Ji X, Guddat LW, Yang K, Rao Z, Yang H. Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2. Proc Natl Acad Sci U S A. 2022 Apr 19;119(16):e2117142119. doi: 10.1073/pnas.2117142119. Epub 2022 Apr 5. PMID: 35380892; PMCID: PMC9172370.
3. Yang H, Rao Z. Structural biology of SARS-CoV-2 and implications for therapeutic development. Nat Rev Microbiol. 2021 Nov;19(11):685-700. doi: 10.1038/s41579-021-00630-8. Epub 2021 Sep 17. PMID: 34535791; PMCID: PMC8447893.