From oncoproteins to spike proteins: the evaluation of intramolecular stability using hydropathic force field

Small protein structural variations can be related to missense mutations generated by single nucleotide polymorphisms, which is the most common human genetic alteration. These mutations, however, occur with different frequencies [19]leading to possibly altered responses to pathogens, chemical agents, or drugs, or altered structural and/or functional characteristics of encoded proteins [20]. Mutations in the Covid-19 Spike glycoprotein are often associated with higher transmissibility, high virulence, and reduced susceptibility to antibody neutralization [21]. Characterizing the stability of thus mutated proteins and their intramolecular connections are important prerequisites for quickly assessing the possible effects of such mutations on the mechanism of action or drug susceptibility.

In order to verify the sensitivity of HINT to estimate these small structural changes, we first applied it to oncoproteins, some of the best-known examples of proteins where altered structures are associated with hyperproliferative developmental disorders and cancer. RAS and RAF are intramolecular kinase proteins that control cellular proliferation, differentiation, and apoptosis [15]. RAS proteins are a small GTPase protein family [22] that exist in two different states: the GTP-bound state (active) and GDP-bound state (inactive) [16]. They possess N, K, and H isoforms; mutations at codons 12, 13, or 61 promote GTP binding and produce constitutive activation of RAS, producing uncontrolled cell proliferation [17]. RAF kinases are a family of three serine/threonine kinases proteins of which B-RAF has the highest basal kinase activity [23]. Sequence analyses of B-RAF genes have identified different mutations in the kinase domain related to this increased kinase activity[18].

All the 18 known mutations of RAS proteins were taken into account and introduced into the wildtype structure of each isoform considering both active and inactive conformations. In total, 76 mutated structures of K- and N-RAS were thus generated. Crystallographic structures of H-RAS have already revealed higher flexibility of the two adjacent regions delimiting the binding pocket known as switch I (residues 32–38) and switch II (residues 60–75) [24]. The first is a single loop while the latter consists of a loop and an α-helix. The active conformation (GTP-bound) shows two possible different orientations of these structural elements, leading to two possible states called state 1 and state 2 in a dynamic equilibrium. Since the recruitment of the effector proteins induces a shift of the conformational equilibrium toward state 2, it is described as the real active state [25]. Considering 18 possible mutations and three different conformations, a total of 57 H-RAS proteins were generated by molecular modeling in our study. Lastly, 18 different B-RAF mutations were introduced into the kinase domain to evaluate their possible effects on intramolecular stability. All mutated structures, after solvation and neutralization, were energy minimized and the Intramolecular HINT scores were calculated for each.

The mutated inactive K- and N-RAS conformations show minimal variations of intramolecular HINT score compared to the wildtype with a not significant ΔHINT score between mutated and wildtype form as shown in Fig. 1. However, these mutations stabilize the active conformations that exhibit higher intramolecular values than the wild type. So, if the intramolecular HINT score of the mutated state is higher than that of the wildtype, the active-inactive conformational equilibrium is shifted towards the active conformation that is more stable than the wildtype (Fig. 1). The stabilization of the closed conformation would thus explain the ability of high-scoring mutations to permanently activate the protein. Also of note is that the mutants with the highest experimental frequency are the most stable (Fig. 1) [16].

Fig. 1figure 1

 K and N-RAS Intramolecular HINT score. K-RAS: The intramolecular stabilities of the mutated inactive (PDB ID: 5W22) and active (PDB ID: 6GOD) conformations of K-RAS were calculated. G12C, G12D, G12V, G13D, G13C, and Q61H are the most stable K-RAS closed conformations and present the highest experimental frequency. H-RAS: The intramolecular stability of the mutated inactive (PDB ID: 6WGH) and active (PDB ID: 5UHV) conformations reveal that G12D, G12S, and Q61R are the most stable and frequent N-RAS closed conformations [16].

Considering the three different known H-RAS conformations, we observed that mutations destabilized the inactive conformations and generated a stabilization of the active state 2 conformations. This result is in line with the explained mechanism of action of H-RAS for which only the state 2 conformation is able to recruit co-activator proteins [25]. According to our calculations, G12S, G12V, G13R, and Q61R are the most stable mutants and present the highest experimental frequency as shown in Fig. 2 [16].

Fig. 2figure 2

 H-RAS structures and Intramolecular HINT score. Three different conformations of H-RAS were identified: an inactive GDP-bound conformation (blue) (PDB ID: 4Q21), an active state 1 conformation (orange) (PDB ID: 3RSO), and an active state 2 conformation (grey) (PDB ID: 5P21). The ΔHINT score between the mutated and the wild-type structure was calculated considering all three possible conformations. Only state 2 active conformation is stabilized by mutations

Although a general stabilization of all RAS mutated active conformation is observed, different mutations produce a different effect on the intramolecular stability due to the nature of the amino-acid substitution and more complex biological mechanism that involve the residues Q61 that show a more significant conformational change when the active conformation dimerize with effector proteins [26].

The intramolecular HINT scores reveal that all active mutated B-RAF proteins are as stable as, or more than, the wildtype. Wan et al. [18] describe the experimental kinase activity of B-RAS mutants. V599D is the registered mutation that most greatly increased kinase activity; our calculations also show that it shows increased protein stability as estimated by the highest intramolecular HINT score value (Fig. 3).

Fig. 3figure 3

B-RAF Intramolecular HINT scores. The ΔHINT score between mutated and wildtype structure (PDB ID: 6XFP) was calculated. All mutated forms are more stable than the wild-type ones. V599D is the most stable and presents the higher experimental kinase activity

Protein stabilization after mutation is often related to local and generally minimal structural variations that include the creation of one or more hydrogen bonds, acquisition of hydrophobic interactions, or reduction of steric bulk. The intramolecular HINT function is sensitive to each of these small changes in the protein’s energetic stability, but also records and accounts for unfavorable interactions such as changes in desolvation energy (unfavorable hydrophobic-polar score) and repulsive Coulombic interactions. In general, the intramolecular HINT function score produces results quite quickly, and these results can be correlated with experimental measurements such as demonstrated above. Also, analysis of the output on a residue pair-by-residue pair basis or even atom-by-atom basis often reveals specific information on the energetics involved in the mutation as shown in Fig. 4.

Fig. 4figure 4

Intramolecular HINT output file. The output file shows each atom-atom interaction and the total intramolecular score with a detailed description of the single energy contribution (hydrogen bond, electrostatic and hydrophobic interactions, and negative contributions). In this capture, for example, the mutated ARG12 establishes two different hydrogen bonds with the residue GLU62.

Sars Cov2 Spike Intramolecular stability

The spike glycoprotein is a well-known and increasingly studied Covid-19 structural protein involved in virus replication due to the interaction between its receptor-binding domain and the human angiotensin-converting enzyme 2 [27]. Several mutations have been identified in different domains that can affect this interaction with the human target or lead to a reduced response to antibodies [28]. Since the spike glycoprotein is one of the main targets of vaccines, antibodies and drugs, understanding the effect of these mutations is necessary to guarantee their long-term efficacy and to monitor the clinical impact of the variant’s diffusion. Previous studies have already demonstrated that protein stability is necessary for survival and diffusion[29] by monitoring the thermodynamic effect of a single mutation on the spike/ACE2 complex stability [30]. Evaluating the effect of a single mutation could be reductive since each variant of concern is characterized by several simultaneous structural mutations. Our aim is to use the intramolecular HINT score to evaluate the stability of the known variants in the closed trimeric conformation of spike glycoprotein, the effect of mutations on the stability of the receptor-binding domain as is, and in complex with the human target.

Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), Gamma (P.1), and Omicron (BA.1) are the principal circulating variants, defined as variants of concern by the U.S. Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/). Their structural characteristics were retrieved from GISAID [31]. Mutations were introduced in the wild-type structures and minimized. The Intramolecular HINT score evaluation shows that all mutated trimeric closed structures are at least as stable as the wildtype and all mutated receptor binding domains (RBDs) are more stable than the wildtype (Fig. 5). Alpha, Beta, and Gamma variants are characterized by one to three mutations in the receptor-binding domain with consequently minimal local variations in the structural stability. The BA.1 (Omicron) variant, despite a huge number of mutations, is stable. The Delta variant’s receptor binding domain presents a lower Intramolecular HINT score than other variants due to L452R mutation, which involves the substitution of a hydrophobic residue (leucine) with a basic and sterically bulky one (arginine) in one of the β-sheets that connect the flexible loop to the domain core.

The RBD-ACE2 complexes are all as or more stable than wildtype ones – thus underlining the variant’s higher affinity toward the human target. This effect is related to the increased local interactions generated by some substitutions like N501Y that characterized the RBD of Alpha, Beta, and Gamma variants, by acquiring a new π-π interaction. The Omicron-ACE2 complex is one of the most stable since the Omicron mutations introduce local positive charges responsible for electrostatic interaction with ACE2, which is negatively charged [32].

Fig. 5figure 5

Intramolecular HINT score of Spike proteins. All mutants generated from the trimeric wildtype closed conformation (PDB ID: 6VXX) are as stable as the wild type. RBD HINT score reveals higher stability of mutants than wildtype. Alpha and Omicron variants present a higher affinity toward ACE2, and their complexes are the most stable

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