Biomedical subjects

P S Brzovic

Publications and source records attributed to P S Brzovic.

8 recordsLinked to original sources

PubMed2001

BRCA1 RING domain cancer-predisposing mutations. Structural consequences and effects on protein-protein interactions.

Cancer-predisposing missense mutations in the RING domain of BRCA1 primarily target Zn(2+)-liganding residues. Here we report on the structural consequences of such mutations introduced into the second Zn(2+) site (Site II) of the BRCA1 RING domain and their effect on the interaction with the BARD1 RING domain. Each of the BRCA1 Site II mutants still interact and form a stable heterodimer with BARD1. Limited proteolysis of BRCA1/BARD1 complexes, monitored by matrix-assisted laser desorption ionization time-of-flight spectrometry, show that the mutations cause a local structural perturbation that is primarily confined to the second Zn(2+) binding loop of the BRCA1 subunit. These findings are consistent with the structure of the BRCA1/BARD1 heterodimer, which shows this region is well removed from the helices required for dimerization with BARD1. Instead, the mutations alter a region of BRCA1 that appears to be required for interaction with ubiquitin-conjugating enzymes.

BRCA1 Protein↗

PubMed2001

Structure of a BRCA1-BARD1 heterodimeric RING-RING complex.

The RING domain of the breast and ovarian cancer tumor suppressor BRCA1 interacts with multiple cognate proteins, including the RING protein BARD1. Proper function of the BRCA1 RING domain is critical, as evidenced by the many cancer-predisposing mutations found within this domain. We present the solution structure of the heterodimer formed between the RING domains of BRCA1 and BARD1. Comparison with the RING homodimer of the V(D)J recombination-activating protein RAG1 reveals the structural diversity of complexes formed by interactions between different RING domains. The BRCA1-BARD1 structure provides a model for its ubiquitin ligase activity, illustrates how the BRCA1 RING domain can be involved in associations with multiple protein partners and provides a framework for understanding cancer-causing mutations at the molecular level.

Amino Acid Sequence↗

PubMed1999

Mapping the functional domains of BRCA1. Interaction of the ring finger domains of BRCA1 and BARD1.

Breast cancer 1 (BRCA1) and BRCA1-associated RING domain 1 (BARD1) are multidomain proteins that interact in vivo via their N-terminal RING finger motif regions. To characterize functional aspects of the BRCA1/BARD1 interaction, we have defined the structural domains required for the interaction, as well as their oligomerization state, relative stability, and possible nucleic acid binding activity. We have found that the RING finger motifs do not themselves constitute stable structural domains but are instead part of larger domains comprising residues 1-109 of BRCA1 and residues 26-119 of BARD1. These domains exist as homodimers and preferentially form a stable heterodimer. Shorter BRCA1 RING finger constructs do not interact with BARD1 or with longer BRCA1 constructs, indicating that the heterodimeric and homodimer interactions are mediated by regions outside the canonical RING finger motif. Nucleic acid binding is a generally proposed function of RING finger domains. We show that neither the homodimers nor the heterodimer displays affinity for nucleic acids, indicating that the proposed roles of BRCA1 and BARD1 in DNA repair and/or transcriptional activation must be mediated either by other regions of the proteins or by additional cofactors.

Amino Acid Sequence↗

PubMed1998

The cancer-predisposing mutation C61G disrupts homodimer formation in the NH2-terminal BRCA1 RING finger domain.

The breast and ovarian cancer tumor suppressor gene, BRCA1, encodes for a Zn2+-binding RING finger motif located near the protein NH2 terminus. The RING finger motif is characterized by eight conserved Cys and His residues which form two Zn2+-binding sites termed Site I and Site II. We used limited proteolysis in conjunction with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy to investigate the metal binding properties and to probe the solution structures of wild-type and mutant BRCA1 constructs that include the RING finger. Our results show that the RING finger motif is part of a larger proteolysis-resistant structural domain which encompasses the first 110 residues of BRCA1. Analytical gel-filtration chromatography and chemical cross-linking experiments demonstrate that the BRCA1 NH2-terminal domain readily homodimerizes in solution. The cancer-predisposing C61G mutation, which alters a conserved Zn2+-binding residue, abolishes metal binding to Site II of the RING finger motif, while Site I remains intact and functional. The C61G mutation also results in increased proteolytic susceptibility of the COOH-terminal portion of the NH2-terminal domain and perturbs the oligomerization properties of BRCA1.

BRCA1 Protein↗

PubMed1998

Ca2+-dependent conformational changes in bovine GCAP-2.

GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent regulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fall in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP-2 is a small N-myristoylated protein containing four EF-hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-binding as measured by a Ca2+-dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP-2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N-terminus of Ca2+-bound GCAP-2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N-terminus of GCAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP-2 and recoverin, the structural consequences of Ca2+-binding for these two proteins are significantly dissimilar.

Amino Acid Sequence↗

PubMed1996

Spectroscopic evidence for preexisting T- and R-state insulin hexamer conformations.

The insulin hexamer is an allosteric protein exhibiting both positive and negative cooperative homotropic interactions and positive cooperative heterotropic interactions (C. R. Bloom et al., J. Mol. Biol. 245, 324-330, 1995). In this study, detailed spectroscopic analyses of the UV/Vis absorbance spectra of the Co(II)-substituted human insulin hexamer and the 1H NMR spectra of the Zn(II)-substituted hexamer have been carried out under a variety of ligation conditions to test the applicability of the sequential (KNF) and the half-site reactivity (SMB) models for allostery. Through spectral decomposition of the characteristic d-->d transitions of the octahedral Co(II)-T-state and tetrahedral Co(II)-R-state species, and analysis of the 1H NMR spectra of T- and R-state species, these studies establish the presence of preexisting T- and R-state protein conformations in the absence of ligands for the phenolic pockets. The demonstration of preexisting R-state species with unoccupied sites is incompatible with the principles upon which the KNF model is based. However, the SMB model requires preexisting T- and R-states. This feature, and the symmetry constraints of the SMB model make it appropriate for describing the allosteric properties of the insulin hexamer.

Allosteric Regulation↗

PubMed1995

Calcium-dependent solvation of the myristoyl group of recoverin.

Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to function as a calcium sensor in visual signal transduction by coupling the kinetics of the recovery phase of the photoresponse to changes in the levels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed that, in the Ca(2+)-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca(2+)-bound conformation, the myristoyl group is exposed to solution. The crystal structure of Ca(2+)-bound recoverin reveals an exposed cluster of hydrophobic residues, raising the possibility that residues in this region may function as part of an intramolecular myristoyl binding site. Fluorescence spectroscopy analysis of interactions between recoverin and 1-anilinonaphthalene-8-sulfonate (ANS) shows that an increase in solvent-accessible hydrophobic surface accompanies Ca2+ binding. 1H nuclear magnetic resonance (NMR) spectra of myristoyl protons show dispersed chemical shifts in the Ca(2+)-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca(2+)-free recoverin show NOE contacts between myristoyl protons and aromatic ring protons. Tryptophan fluorescence quenching by acrylamide indicates that the myristoyl group is in proximity to a tryptophan residue only in the Ca(2+)-free conformation. These results indicate that the myristoyl group is in contact with residues in the hydrophobic cluster in Ca(2+)-free recoverin and that it is exposed to solution in the Ca(2+)-bound conformation.

Anilino Naphthalenesulfonates↗

PubMed1995

Ligand binding to wild-type and E-B13Q mutant insulins: a three-state allosteric model system showing half-site reactivity.

By using ultra-violet and visible absorbance in conjunction with high field 1H-nuclear magnetic resonance spectroscopy, the insulin hexamer has been shown to undergo two allosteric transitions in solution involving three allosteric states (T6<-->T3 R3<-->R6). A simple mathematical model consisting of four variables has been derived that quantitatively describes the complex homotropic and heterotropic interactions that modulate these allosteric transitions. The mutation of one residue, Glu-B13 to Gln, results in an unexpected change in the T3R3 to R6 equilibrium by a factor of 10(7).

Allosteric Site↗