Ravi Basavappa

The regions of securin and cyclin B proteins recognized by the ubiquitination machinery are natively unfolded

The proteins securin and cyclin B are destroyed in mitosis by the ubiquitin/proteasome system. This destruction is important to mitotic progression. The N‐terminal regions of these proteins contain the sequence features recognized by the ubiquitination system. We have demonstrated using circular dichroism and 1‐D and 2‐D nuclear magnetic resonance that these rather substantial regions are natively unfolded. Based on these findings, we propose a model that helps to explain previously enigmatic observations.

The crystal structure of human cyclin B.

Cyclin B is the key regulatory protein controlling mitosis in all eukaryotes, where it binds cyclin-dependent kinase, cdk1, forming a complex which initiates the mitotic program through phosphorylation of select proteins. Cyclin B regulates the activation, subcellular localization, and substrate specificity of cdk1, and destruction of cyclin B is necessary for mitotic exit. Overexpression of human cyclin B1 has been found in numerous cancers and has been associated with tumor aggressiveness. Here we report the crystal structure of human cyclin B1 to 2.9 Å. Comparison of the structure with cyclin A and cyclin E reveals remarkably similar N-terminal cyclin box motifs but significant differences among the C-terminal cyclin box lobes. Divergence in sequence gives rise to unique interaction surfaces at the proposed cyclin B/ cdk1 interface as well as the ‘RxL’ motif substrate binding site on cyclin B. Examination of the structure provides insight into the molecular basis for differential affinities of protein based cyclin/cdk inhibitors such as p27, substrate recognition, and cdk interaction.

Isolation and Characterization of Androgen Receptor Mutant, AR(M749L), with Hypersensitivity to 17-β Estradiol Treatment

Estrogens, primarily 17β-estradiol (E2), may play important roles in male physiology via the androgen receptor (AR). It has already been shown that E2 modulates AR function in LNCaP prostate cancer cells and xenograft CWR22 prostate cancer tissues. Using a molecular model of E2 bound-AR-ligand binding domain (LBD) and employing site-directed mutagenesis strategies, we screened several AR mutants that were mutated at E2-AR contact sites. We found a mutation at amino acid 749, AR(M749L), which confers AR hypersensitivity to E2. The reporter assays demonstrate that E2 can function, like androgen, to induce AR(M749L) transactivation. This E2-induced AR mutant transactivation is a direct effect of the AR(M749L), because the transactivation was blocked by antiandrogens. The hypersensitivity of AR(M749L) to E2 is not due to increased affinity of AR(M749L) for E2, rather it may be due to the existence of the proper conformation necessary to maintain E2 binding to the AR-LBD long enough to result in E2-induced transactivation. AR(M749L) transactivation can be further enhanced in the presence of AR coregulators, such as ARA70 and SRC-1. Therefore, amino acid 749 may represent an important site within the AR-LBD that is involved in interaction with E2 that, when mutated, allows E2 induction of AR transactivation.

15N Relaxation Studies of Apo-Mts1: A Dynamic S100 Protein†

Mts1 is a member of the S100 family of EF-hand calcium-binding proteins. Like most S100 proteins, Mts1 exists as a dimer in solution and contains one canonical and one pseudo-EF-hand motif per monomer, each of which consists of two alpha helices connected by a loop capable of coordinating a calcium ion. The backbone dynamics of murine apo-Mts1 homodimer have been examined by nuclear magnetic resonance spectroscopy. Longitudinal and transverse relaxation data and steady-state (1)H- (15)N nuclear Overhauser effects were analyzed using model-free formalism. The extracted global correlation time is 9.94 ns. Results indicate that the protein backbone is most rigid at the dimer interface, made up of helices 1 and 4 from each monomer with mean S (2) ( S avg (2)) values approximately 0.9, flanked by helices 2 and 3 with lower S avg (2) values of 0.84 and 0.77, respectively. Each calcium-binding site along with the hinge joining the two EF-hands and the N- and C-termini are considerably more flexible than the dimer interface on a range of time scales and more flexible than the corresponding regions of other S100 proteins studied to date. As the hinge and the C-terminal tail are believed to interact with target proteins, these dynamic characteristics may have implications for Mts1 activity.

A quick and gentle method for mounting crystals in capillaries

The capillary mounting of protein crystals is still necessary under some circumstances, despite its being supplanted by loop mounting/flash freezing as the favored method for crystal mounting prior to data collection. Traditional capillary mounting methods require a degree of dexterity and finesse that causes apprehension among many crystallographers. Here a simple method for capillary mounting that uses a loop for depositing the crystal in the capillary is described.

Letter to the Editor: Sequence-specific chemical shift assignment and chemical shift indexing of murine apo-Mts1

Members of the S100 family of calcium-binding proteins have been linked to a wide variety of cellular functions, including cell growth, differentiation, cytoskeletal dynamics, inflammation and calcium homeostasis. S100A4, also known as Mts1, has been linked to metastatic capabilities of cells (Ebralidze et al., 1989; Davies et al., 1993; Grigorian et al., 1993; for reviews see Barraclough, 1998; Sherbet and Lakshmi, 1998) through several lines of evidence including: (1) Mts1 RNA is transcribed at detectable levels only in metastatic (and blood) cancers, and not in nonmetastatic cancers; (2) introduction of Mts1 into nonmetastatic cell lines derived from mammary tumors confers the metastatic phenotype; (3) cross breeding of mice prone to non-metastatic tumors with Mts1 transgenic mice results in offspring with substantially increased aggressiveness of tumors; (4) use of antisense ribozyme techniques to reduce available Mts1 RNA levels diminishes metastatic character of tumor cells. As with other S100 proteins, calcium binding is believed to trigger a conformational switch which is critical for Mts1 function. Here, we present the sequential heteronuclear chemical shift assignments of Mts1 in its calcium free form.

Letter to the Editor: Sequence-specific chemical shift assignment of calcium-loaded murine S100A4

S100A4, also known as Mts1, has been linked to metastasis through several lines of evidence (Ebralidze et al., 1989; Davies et al., 1993; Grigorian et al., 1993; for reviews see Barraclough, 1998; Sherbet and Lakshmi, 1998). Mts1 expression levels are higher in metastatic than in non-metastatic tumors, and are also high in cells with naturally high motility. Introduction of Mts1 can increase the metastatic character of cancer cells, while antisense ribozyme techniques targeted to Mts1 can reduce metastasis. Cross-breeding of mice engineered to express high levels of Mts1 with mice highly susceptible to non-metastatic tumors results in offspring with more aggressive tumors. Mts1 belongs to the S100 protein family, most members of which contain two calcium-coordinating EF hands. Calcium binding triggers a conformational rearrangement which can alter surfaces required for interaction with other proteins. Here, we present the sequential heteronuclear chemical shift assignments of Mts1 in its calcium-loaded form.