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The impact of oncogenic mutations of the viral Src kinase on the structure and stability of the SH3 domain - PubMed

  • ️Fri Jan 01 2021

The impact of oncogenic mutations of the viral Src kinase on the structure and stability of the SH3 domain

M Carmen Salinas-Garcia et al. Acta Crystallogr D Struct Biol. 2021.

Abstract

Src kinase belongs to the family of Src-related nonreceptor tyrosine kinases. Because of its physiological role in cell growth and proliferation, its activity is strictly controlled by several mechanisms. Nevertheless, in viral Src kinase (v-Src) some of these mechanisms fail, and its uncontrolled activity is responsible for the occurrence of cancer. Here, the crystal structures of three SH3-domain mutants of v-Src were determined to unveil the effects of these oncogenic mutations in this regulatory domain. Mutations in the n-Src and distal loops have a low impact on the overall structure of the domain and its capacity to form intertwined dimers. However, mutations in the RT loop compromise the stability of the domain and make the protein very prone to aggregation. Additionally, these mutations prevent the formation of intertwined dimers. The results show a synergistic effect between mutations in the RT loop and those in the n-Src and distal loops. Analysis of the structures of the v-Src SH3-domain mutants and the closed inactive conformation of cellular Src kinase (c-Src) point to a loss of the interactions that are required to establish the compact inactive form of the kinase. Nevertheless, an analysis of structures of the c-Src SH3 domain complexed with class I and II peptides points to minor changes in the interactions between the v-Src SH3 domain and these peptides. In this way, the structures reported here indicate that mutations in the RT loop might impair the kinase regulation mechanism without affecting the recognition of short proline-rich motifs in the target proteins of the kinase, thus explaining the oncogenic behaviour of the protein.

Keywords: SH3 domain; Src kinase; crystal structure; intertwined dimer; oncogens; tyrosine kinases.

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Figures

Figure 1
Figure 1

Sequences of the SH3-domain fragment of chicken c-Src and the mutants of the v-Src SH3 domain studied in this work. The v-Src residues (Schmidt Ruppin E strain; UniProt entry P00524) are shown in bold.

Figure 2
Figure 2

(a) Intertwined dimer of the v-Src SH3 W95R-I96T variant (PDB entry

7net

; chain A, blue; chain B, yellow) overlaid on the c-Src SH3-domain open protomer (chain A, grey; PDB entry

6xvo

). The average backbone r.m.s.d. value is 0.28 Å. Mutated residues in the n-Src and distal loops are shown as sticks (chain A). (b) Superposition of the v-Src SH3 W95R-I96T variant intertwined dimer (chain A, blue; chain B, yellow) and the c-Src SH3-domain monomer (chain A, purple; chain B, cyan; PDB entry

6xvo

). (c) Residues in the distal loop are shown as sticks.

Figure 3
Figure 3

DLS measurements of the v-Src SH3 variants. (a) v-Src SH3 W95R-I96T at 5 mg ml−1 in 50 mM sodium acetate buffer pH 5.0 in the absence (red line) and in the presence (black line) of 5% PEG 300 at 25°C. In the absence of PEG, the protein is a monomer with R h = 1.8 ± 0.3 nm. After adding 5% PEG 300, the hydrodynamic radius increases to a value of 2.4 ± 0.5 nm. (b) Temperature-dependent aggregation of the v-Src SH3 N117D-V124L variant. After incubation for 24 h at three different temperatures, the protein was measured at 5 mg ml−1 in 50 mM sodium phosphate buffer pH 7.0. At low temperature (≤15°C) 99.9% of the protein is a monomer with an R h of 1.8 ± 0.4 nm. At higher temperatures, the protein aggregates, forming high-molecular-weight oligomers, and after one day of incubation the monomer population decreases at both 20°C (66%) and 25°C (39%). These oligomers tested positive for amyloid aggregates using the ThT and CR assays.

Figure 4
Figure 4

Cartoon representation of the superposition of the c-Src SH3 domain (PDB entry

6xvn

; chain A, purple; chain B, cyan) and (a) the v-Src SH3 N117D-V124L variant (PDB entry

7nes

) and (b) the v-Src SH3 Q128R variant (PDB entry

7ner

). In both chains of the c-Src SH3 domain, the n-­Src loop has been partially modelled and the unmodelled residues are represented by a dashed line. Hydrogen bonds in the RT loop of (c) the v-­Src SH3 N117D-V124L variant, (d) the v-Src SH3 Q128R variant and (e) the c-Src SH3 domain (chain A).

Figure 5
Figure 5

Native fraction of the v-Src SH3 mutants versus pH at 25°C: v-Src SH3 Q128R (blue circles and line), v-Src SH3 W95R-I96T (green triangles and dashed line) and v-­Src SH3 N117D-V124L (red squares and dashed/dotted line). For comparison, data for c-Src SH3 have also been included (black diamonds and dotted line).

Figure 6
Figure 6

Native fraction of v-Src SH3 mutants versus GndHCl concentration at pH 7.0 and 25°C: v-Src SH3 Q128R (blue circles and line), v-Src SH3 W95R-I96T (green triangles and dashed line) and v-Src SH3 N117D-V124L (red squares and dashed/dotted line). For comparison, the unfolding curve of c-Src SH3 (black diamonds and dotted line) has also been included.

Figure 7
Figure 7

Interactions at the interface of the intertwined dimer of v-Src SH3 W95R-I96T (PDB entry

7net

). The residues in the different chains of the dimer are coloured grey (chain A) and green (chain B). The structure of the v-­Src SH3 Q128R mutant (yellow; PDB entry

7ner

) is overlaid on one of the dimer chains. The mutated residues, Trp95 and Ile96, are shown as sticks. The Thr96(A)/Thr98(A) residues form a hydrogen bond mediated by a water molecule (W) to the symmetry-related Thr96(B)/Thr98(B) residues. For the sake of clarity, these symmetry-related residues are not represented. Asp99 show a double conformation, which facilitates intra-chain hydrogen bonds with different residues in the RT loop.

Figure 8
Figure 8

Superposition of the (a) v-Src SH3 Q128R (green) and (b) v-Src SH3 N117D-V124L (yellow) variants with the structure of the c-Src SH3 Q128R–APP12 complex (PDB entry

5ob1

). The SH3 domain is represented as a cartoon (white) and the peptide as sticks (magenta). The mutations present in each oncogenic variant are represented as sticks. The Asp99–APP12–Arg7 salt bridge that drives the peptide orientation upon binding is also shown.

Figure 9
Figure 9

(a) Interactions between the SH2–kinase linker (grey) and the SH3 domain (green). Residues of the SH2 and kinase domains that also interact with the SH3 domain in the structure of the c-Src kinase (PDB entry

2ptk

) are shown as orange sticks. The interactions between residues in the SH3 domain and SH2–kinase linker (shown as sticks) are marked by green dashed lines. The three interaction pockets are marked in orange and labelled P1, P2 and P3. (b) Superposition of the SH3 domain of the c-Src kinase and the v-Src SH3 Q128R mutant (PDB entry

7ner

, yellow, cartoon). The oncogenic mutations Trp95 and Asn117 are shown as yellow sticks and the interactions established by Arg95 and Asp117 have been removed. (c) Closed inactive conformation of the c-Src tyrosine kinase from chicken (PDB entry

2ptk

). The SH1-domain N- and C-­terminal lobes are coloured magenta and pink, respectively. The SH2 domain is coloured blue and the SH2–SH1 linker cyan. The SH3 domain is shown in green and the superposition of v-Src SH3 is shown in yellow. The mutated residues in the SH3 domain are shown as sticks.

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