Table 1.

Structure-based analysis of BRCA1 variants in the BRCT domains

ClassVariantNotes
Hydrophobic core disruptionG1706EG1706 in BRCT-N is completely inaccessible to solvent. This position is in the α2 helix and sits between two positively charged Lys residues, one of which (K1702) forms hydrogen bonds with SEP6, a phosphorylated Ser critical to BRCA1 phosphopeptide binding. The substitution by Glu introduces a buried negative charge, which is energetically unfavorable.
V1713AV1713 lies in the β4 strand of the four-stranded parallel β-sheet in BRCT-N. In BRCT tandem domains, hydrophobic, β-branched amino acid residues are conserved at this position and are probably critical to the stability of the BRCT tandem domain fold. β-branched amino acid residues are generally favored in β sheets and the Ala, although hydrophobic, is not β-branched.
V1736AV1736 is completely buried in a tightly packed neighborhood, rich in hydrophobic residues and methyl(ene) groups. The Ala replacement results in loss of a single methyl group and creation of a small cavity in this neighborhood. The destabilizing effects of such cavities have been shown to be correlated with the number of methyl(ene) groups within 6 Å of a mutated position ( 44), so this cavity is likely destabilizing. Interestingly, both valines in the conserved W1712VVS1715 motif, critical for the stability of the BRCT fold, are within 4 Å of this position.
G1738R, G1738EG1738 is in the linker region connecting the two BRCT domains and is completely inaccessible to solvent. Our structural modeling indicates that introduction of either an Arg or Glu at this position creates numerous steric clashes. Both Arg and Glu are charged amino acid residues, and placement of a charged residue in a buried position is expected to be destabilizing.
I1766SI1766 is completely buried in the hydrophobic core of BRCT-C. The replacement by the polar serine introduces a side chain oxygen atom (OG) that can function as a hydrogen bond donor or acceptor. Inspection of the local environment indicates that there are no available donor or acceptor atoms to satisfy the H-bonding potential of the OG, yielding a destabilized structure.
Phosphopeptide binding disruptionS1655FS1655 is a critical residue for phospho-Ser recognition, and forms hydrogen bonds with the phospho-Ser pSer0 of CtIP peptide in the BRCA BRCT tandem domain X-ray crystal structure 1y98 ( 45) and with the phospho-Ser pSer6 of BACH1 peptide in 1t29 and 1t2v ( 41). Replacement of Ser by Phe breaks hydrogen bonds between S1655 and pSer and also creates steric clashes between pSer and the phenyl ring in all of these structures.
T1700AT1700 is part of a hydrogen bonding network in the holo structures of the BRCA1 BRCT domains in complex with BACH1 peptide [PDB 1t29 and 1t2v] ( 23, 41) and in complex with CtIP peptide [PDB 1y98] ( 45). The H-bond network includes the phosphorylated Ser (SEP6) of the BACH1 and CtIP, identified as a critical residue in phosphopeptide binding to BRCA1. In these structures, the side chain oxygen OG1 of T1700 forms a H-bond with the side chain OG1 oxygen of S1655, which in turn forms a H-bond with the phosphate oxygen O1P in the phosphorylated Ser SEP6.
L1764PL1764 is in the β1 strand of BRCT-C. We predict fold destabilization upon Pro substitution because this position is a key hydrophobic residue in the BRCT superfamily ( 2) and Pro is not hydrophobic. Furthermore, Pro is compatible with a limited number of backbone conformations, preferring a PHI backbone dihedral angle close to −60 deg. Position 1,764 has a PHI angle of −112 deg that is not compatible with Pro.
Disruption of putative binding sitesR1753TR1753 is in the linker region that connects the two BRCT domains. The substitution by Thr results in loss of a positively charged side chain in an exposed position. Here, the position occurs in a surface patch highly conserved among BRCA1 orthologues from mammals through Xenopus and Tetraodon. The surface electrostatic potential, as calculated by DELPHI using united atom AMBER charges, is substantially different for the Arg and Thr variants. The substitution produces an acidic patch on the surface where the Thr is exposed and observable changes in electrostatic potential around neighboring exposed residues in the linker region. The high conservation in this region may be the signature of a BRCA1-specific binding site with a protein partner. Interestingly, the linker region in the 53BP1 BRCT dimer has been identified as a binding site of p53 ( 46, 47).
No evidence for functional impactL1664PL1664 lies at the second turn of an α-helix in BRCT-N (helix α1). In general, Pro amino acid residues past the first turn of a helix are destabilizing because of steric clashes with backbone atoms. However, although helix α1 may be destabilized by P1664, the results of our assay indicate that the effect is not strong enough to disrupt the hydrophobic core of the protein. The Pro side chain bonds to a backbone nitrogen atom and is only compatible with a limited number of backbone conformations, preferring a PHI backbone dihedral angle close to −60 deg. Here, position 1,664 has a PHI angle of −57.5 deg that is compatible with Pro.
Q1785HQ1785 in BRCT-C is on a protruding area of the protein surface that is not conserved among BRCA1 orthologues. It lies on the exposed, hydrophilic side of the amphipathic helix α1′ and sticks out into solvent. Although the substitution with His breaks a possible H-bond between the NE2 nitrogen of the Gln and the backbone carbonyl oxygen of Q1781, the Q1781 acceptor can form a compensating H-bond with solvent, or possibly the backbone nitrogen of Q1785.
E1794DE1794 in BRCT-C sticks out into solvent. Substitution by Asp, another negatively charged residue, is not likely to have any functional effect.
V1804DV1804 in BRCT-C is on a flexible loop and sticks out into solvent. Introduction of a charged residue on the surface is not likely to affect function unless it is at a binding site. This position is probably not a binding site because it lies on a surface region that is not conserved among BRCA1 orthologues.
P1806AP1806 in BRCT-C is found in β-strand β3 and is solvent exposed. Substitution by Ala is not likely to have any functional effect. This position is probably not a binding site because it lies in a surface region that is not conserved among BRCA1 orthologues.