Thermal unfolding of human BRCA1 BRCT-domain variants

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Abstract

Missense mutations at the BRCT domain of human BRCA1 protein have been associated with an elevated risk for hereditary breast/ovarian cancer. They have been shown to affect the binding site and they have also been proposed to affect domain stability, severely hampering the protein's tumor suppressor function. In order to assess the impact of various such mutations upon the stability and the function of the BRCT domain, heat-induced denaturation has been employed to study the thermal unfolding of variants M1775R and R1699W, which have been linked with the disease, as well as of V1833M, which has been reported for patients with a family history. Calorimetric and circular dichroism results reveal that in pH 9.0, 5 mM borate buffer, 200 mM NaCl, analogously to wild type BRCT, all three variants undergo partial thermal unfolding to a denatured state, which retains most of the native's structural characteristics. With respect to wild-type BRCT, the mutation M1775R induces the most severe effects especially upon the thermostability, while R1699W also has a strong impact. On the other hand, the thermal unfolding of variant V1833M is only moderately affected relative to wild-type BRCT. Moreover, isothermal titration calorimetric measurements reveal that contrary to M1775R and R1699W variants, V1833M binds to BACH1 and CtIP phosphopeptides.

Introduction

A large number of germ-line mutations in the human BRCA1 gene have been linked with inherited breast and ovarian cancer [1], [2]. Most are frame shift or nonsense mutations, which can straightforwardly be assigned as deleterious since they lead to truncated proteins. On the other hand, over one hundred missense mutations located in the BRCT domain, detected from genetic screening worldwide, have been deposited at the Breast Cancer Information Core (BIC) as unclassified variants. In vitro functional studies have shown that quite a few can cause loss of the protein's function [3], [4], [5], [6], while co segregation analysis has revealed the association of a handful of these missense mutations to hereditary breast/ovarian cancer.

The crystal structures of the tandem BRCT repeats (each containing ∼ 90 aminoacids) comprising the C-terminal functional BRCT domain of BRCA1 [7] as well as of 53BP1 [8], reveal a common structural motif, which is highly conserved among BRCT repeats. It consists of a parallel four-stranded β-sheet located at the central part of the fold surrounded by three α-helices. The two BRCT repeats pack together in a specific head-to-tail manner, giving rise to the formation of a conserved, almost all-hydrophobic inter-repeat interface. A flexible, BRCT-repeat-interconnecting, 23-residue linker is also involved in the interface formation since the α-helix at its center packs right against the inter-BRCT interface. The structural integrity of the tandem repeats is essential for the molecule's interaction with phosphorylated protein targets [9] including the DNA helicase BACH1 and the CtBP-interacting protein CtIP [10]. The binding of the BRCA1 BRCT domain to BACH1 and the transcriptional corepressor CtIP plays an important role in the control of the G2/M phase checkpoint [11], [12]. The crystal structure of the BRCT complex with the phosphopeptides containing the sequence pSer-X-X-Phe, (phosphorylation of Ser990 and Ser327 for the BACH1 and CtIP phosphopetides respectively, X stands for any residue [13], [14], [15], [16]) revealed that phosphoserine binds at a conserved pocket at the N-terminal BRCT repeat while the hydrophobic groove at the inter-BRCT interface region recognizes phenylalanine. A higher affinity of the BRCT domain has been shown for BACH1 than CtIP due to the interactions of the neighboring aminoacids [16]. Missense mutations at the BRCT domain have been proposed to induce the molecule's loss of function by directly altering the binding sites and/or by destabilization. Characteristically, NMR structural results of the BRCT-domain suggest that for biologically active conformations the proper alignment of the N and C-terminal BRCT repeats is essential [17].

The cancer-linked BRCT missense mutation M1775R cripples the DNA repair and transcription function of BRCA1 [18], [19] and inhibits BRCT interactions with the DNA helicase BACH1 [20] as well as with the transcriptional co-repressor CtIP [21], [22]. Met1775 is located at the inter-BRCT-repeat interface. It is well conserved among the available BRCA1 BRCT domain orthologs. The crystal structure of the M1775R mutant revealed that the methionine–arginine substitution leads to a disruption of the hydrogen-bond network at the inter-BRCT-repeat interface [23]. Direct comparisons with the crystallographic studies of the BRCT-phosphopeptide complex [15] demonstrated that Arg1775, blocks access of the peptide-Phe(+3) (Phe993 for BACH1 and Phe330 for CtIP) to the hydrophobic inter-BRCT-repeat groove.

Residue Arg1699 is highly conserved and it too is located at the inter-BRCT-repeat interface region. It participates in the formation of a salt bridge between the N and C-terminal BRCT repeats. The arginine to tryptophan substitution leads to the loss of salt-bridging interactions while inducing steric strain associated with the accommodation of the tryptophan residue [23]. Moreover, Arg1699 is part of the sides of the pocket recognizing the Phe(+ 3) peptide residue, playing an important role in the alignment of the main chain of Phe(+ 3) [15].

The variant V1833M has only been reported in BIC three times, one of which concerns a Greek family with two ovarian cancer incidents [24]. Val1833 is highly conserved and although the substitution to methionine is predicted to be a mild one [3], a possible destabilization of the overall fold has been suggested [25] since Val1833 is located in the hydrophobic core of the C-terminal BRCT repeat. Interestingly, chemical denaturation experiments have shown that the valine to methionine substitution reduces the thermodynamic stability of the BRCT domain with respect to an intermediate fold, exhibiting an almost complete loss of structural features, by approximately 5.5 kcal/mol [26]. Destabilization of the overall fold has also been proposed for A1708E located right in the hydrophobic inter-repeat interface and G1738R at the inter-repeat linker.

Thermal denaturation experiments of the BRCT domain of human BRCA1 have shown that the unfolding of the protein occurs via an aggregation-prone partly unfolded denatured state that is structurally very similar to the native [27]. Here we present thermal unfolding results for variants M1775R, R1699W, and V1833M obtained via high-sensitivity differential scanning calorimetry (DSC) and circular dichroism (CD). For all three variants we show that heat-induced denaturation always involves a partly unfolded, denatured state, which retains most of the native's secondary structural characteristics. The three mutations have varying effects upon the molecule's thermostability with respect to wild type, depending on their location relative to the hydrophobic inter-BRCT-repeat interface. Moreover, isothermal titration calorimetric studies demonstrate that contrary to M1775R and R1699W variants, V1833M binds to both the BACH1 and the CtIP phosphopeptides.

Section snippets

Gene subcloning and mutagenesis

The encoding region for human wild type and mutant A1708E, BRCA1 BRCT-tan domain (a.a. 1646–1859) were amplified by PCR from plasmids kindly provided by Prof. A.N. Monteiro (Cornell University), using the following primers: (i) BRCT (NdeI), ACATATGGTCAACAAAAGAATGTGCATGGTG and (ii) BRCT (BamHI), AGGATCCTCAGGGGATCTGGGGTATCAGG with NdeI and BamHI restriction sites at their 5′ and 3′ ends, respectively. The resulting 750 bp PCR products were ligated into pCR2.1 cloning vector and transformed into

Results

The BRCT domain variants R1699W, M1775R and V1833M were expressed in E. coli. They were obtained in highly soluble form, more than 99% pure and stable for several days at 4 °C. SDS-PAGE showed the presence of a single band at the expected MW ∼ 25 kDa. The three mutated residues are shown in the BRCT structure presented in Fig. 1 [7]. While Met1775 and Arg1699 are both located at the hydrophobic interface between the two BRCT repeats formed by the packing of three helices: α2 (of the N-terminal

Discussion

For all the samples studied by DSC, the obtained per-residue values of the calorimetric parameters (ΔH)cal and ΔCp are small compared to globular proteins of same size [32], [33]. This is indicative that limited structural changes take place during the observed thermal denaturation process. For wt-BRCT as well as for the three variants the per-residue values of (ΔH)cal varied between 220 and 400 cal/mol-residue and ΔCp varied between 2.2 and 3.0 cal/K mol-residue. In fact, additional thermal

Acknowledgements

G.N., S.P., and A.T. acknowledge support from the Graduate Fellowship Program of NCSR “Demokritos”. We would like to thank Prof. A. Monteiro for kindly providing the BRCT plasmids. The use of the CD facilities at the Centre for Crystallographic Studies of Macromolecules of NCSR “Demokritos” is acknowledged. This work was supported by funding from the General Secretariat of Research and Technology of Greece, Excellence in Research II, funded by 75% from the European Union.

References (38)

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