BRCA1 is a tumor suppressor gene that is mutated in families with breast and ovarian cancer. Several BRCA1 splice variants are found in different tissues, but their subcellular localization and functions are poorly understood at the moment. We previously described BRCA1 splice variant BRCA1a to induce apoptosis and function as a tumor suppressor of triple negative breast, ovarian and prostate cancers. In this study we have analyzed the function of BRCA1 isoforms (BRCA1a and BRCA1b) and compared them to the wild type BRCA1 protein using several criteria like studying expression in normal and tumor cells by RNase protection assays, sub cellular localization/fractionation by immunofluorescence microscopy and western blot analysis, transcription regulation of biological relevant proteins and growth suppression in breast cancer cells. We are demonstrating for the first time that ectopically expressed GFP-tagged BRCA1, BRCA1a, and BRCA1b proteins are localized to the mitochondria, repress ELK-1 transcriptional activity and possess antiproliferative activity on breast cancer cells. These results suggest that the exon 9,10 and 11 sequences (aa 263 – 1365) which contain two nuclear localization signals, p53, Rb, c-Myc, γ- tubulin, Stat, Rad 51, Rad 50 binding domains, angiopoietin-1 repression domain are not absolutely required for mitochondrial localization and growth suppressor function of these proteins. Since mitochondrial dysfunction is a hallmark of cancer, we can speculate that the mitochondrial localization of BRCA1 proteins may be functionally significant in regulating both the mitochondrial DNA damage as well as apoptotic activity of BRCA1 proteins and mislocalization causes cancer.

Ca2+ controls biological processes by interacting with proteins with different affinities, which are largely influenced by the electrostatic interaction from the local negatively charged ligand residues in the coordination sphere. We have developed a general strategy for rationally designing stable Ca2+- and Ln3+-binding proteins that retain the native folding of the host protein. Domain 1 of cluster differentiation 2 (CD2) is the host for the two designed proteins in this study. We investigate the effect of local charge on Ca2+-binding affinity based on the folding properties and metal-binding affinities of the two proteins that have similarly located Ca2+-binding sites with two shared ligand positions. While mutation and Ca2+ binding do not alter the native structure of the protein, Ca2+ binding specifically induced changes around the designed Ca2+-binding site. The designed protein with a -5 charge at the binding sphere displays a 14-, 20-, and 12-fold increase in the binding affinity for Ca2+, Tb3+, and La3+, respectively, compared to the designed protein with a -3 charge, which suggests that higher local charges are preferred for both Ca2+ and Ln3+ binding. The localized charged residues significantly decrease the thermal stability of the designed protein with a -5 charge, which has a T(m) of 41 degrees C. Wild-type CD2 has a T(m) of 61 degrees C, which is similar to the designed protein with a -3 charge. This decrease is partially restored by Ca2+ binding. The effect on the protein stability is modulated by the environment and the secondary structure locations of the charged mutations. Our study demonstrates the capability and power of protein design in unveiling key determinants to Ca2+-binding affinity without the complexities of the global conformational changes, cooperativity, and multibinding process found in most natural Ca2+-binding proteins.

Electrostatic interactions are important for molecular recognition processes including Ca 2+ -binding and cell adhesion. To understand these processes, we have successfully introduced a novel Ca 2+ -binding site into the non-Ca 2+ -dependent cell adhesion protein CD2 using our criteria that are specifically tailored to the structural and functional properties of the protein environment and charged adhesion surface. This designed site with ligand residues exclusively from the b-sheets selectively binds to Ca 2+ and Ln 3+ over other mono-and divalent cations. While Ca 2+ and Ln 3+ binding specifically alters the local environment of the designed Ca 2+ -binding site, the designed protein undergoes a significantly smaller conformation change compared with those observed in naturally occurring Ca 2+ -binding sites that are composed of at least part of the flexible loop and helical regions. In addition, the CD2-CD48-binding affinity increased approximately threefold after protein engineering, suggesting that the cell adhesion of CD2 can be modulated by altering the local electrostatic environment. The study provides site-specific information for regulating cell adhesion within CD2 and gives insight into the structural factors required for Ca 2+ -modulated biological processes.

Ca2+ binding proteins are essential for regulating the role of Ca2+ in cell signaling and maintaining Ca2+ homeostasis. Negatively charged residues such as Asp and Glu are often found in Ca2+ binding proteins and are known to influence Ca2+ binding affinity and protein stability. In this paper, we report a systematic investigation of the role of local charge number and type of coordination residues in Ca2+ binding and protein stability using de novo designed Ca2+ binding proteins. The approach of de novo design was chosen to avoid the complications of cooperative binding and Ca2+-induced conformational change associated with natural proteins. We show that when the number of negatively charged coordination residues increased from 2 to 5 in a relatively restricted Ca2+-binding site, Ca2+ binding affinities increased by more than 3 orders of magnitude and metal selectivity for trivalent Ln3+ over divalent Ca2+ increased by more than 100-fold. Additionally, the thermal transition temperatures of the apo forms of the designed proteins decreased due to charge repulsion at the Ca2+ binding pocket. The thermal stability of the proteins was regained upon Ca2+ and Ln3+ binding to the designed Ca2+ binding pocket. We therefore observe a striking tradeoff between Ca2+/Ln3+ affinity and protein stability when the net charge of the coordination residues is varied. Our study has strong implications for understanding and predicting Ca2+-conferred thermal stabilization of natural Ca2+ binding proteins as well as for designing novel metalloproteins with tunable Ca2+ and Ln3+ binding affinity and selectivity.PACS codes: 05.10.-a

Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+‐induced conformational changes. Investigation into the determinants for Ca2+‐induced conformational change is often impeded by cooperativity between multiple metal‐binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+‐dependent conformational changes, we report the design of a single‐site Ca2+‐binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2). CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 ± 0.1 and 90 ± 25 μm, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native‐like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+‐dependent proteins and coupled metal‐binding sites, represents an important step in the design of trigger proteins.