Purpose: To review the recent advances in the atomic-level understanding of the epidermal growth factor receptor (EGFR) tyrosine kinase (TK). We aim to highlight the current and future importance of these studies for the understanding and treatment of malignancies where EGFR-TK is improperly activated. Methods: The analysis was conducted on published crystal structures deposited in the Protein Data Bank ( www.pdb.org ) using the program O. Results: In this review we emphasize how recent EGFR kinase domain crystal structures can explain the mechanisms of activation for L858R and other EGFR-TK mutations, and compare these distinct activating mechanisms with those recently described for the wild-type EGFR. We suggest an atomic-level mechanism for the poor efficacy of lapatinib against tumors with activating EGFR kinase domain point mutations compared with the efficacy of gefitinib and erlotinib, and demonstrate how structural insights help our understanding of acquired resistance to these agents. We also highlight how these new molecular-level structural data are expected to affect the development of EGFR-TK targeted small molecule kinase inhibitors. Conclusion: There are now more crystal structures published for the EGFR-TK domain than for any other TK. This wealth of crystallographic information is beginning to describe the mechanisms by which proper regulation of EGFR-TK is lost in disease. These crystal structures are beginning to show how small molecules inhibit EGFR-TK activity and will aid development of EGFR-TK mutant targeted therapies.

The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca ²⁺ -permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720–797), a flexible linker (798–827), and an oligomeric coiled coil domain (828–895). We found that PC2-EF binds Ca ²⁺ at a single site and undergoes Ca ²⁺ -dependent conformational changes, suggesting it is an essential element of Ca ²⁺ -sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca ²⁺ -bound PC2-EF. Human PC2-EF contains a divergent non-Ca ²⁺ -binding helix-loop-helix (HLH) motif packed against a canonical Ca ²⁺ -binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R ₁ and R ₂ relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca ²⁺ -bound states, consistent with a role for PC2-EF as a Ca ²⁺ -sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca ²⁺ -dependent protein interactions. We propose that Ca ²⁺ -sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca ²⁺ dependence of PC2 channel activity by PC2-EF.

Polycystin-2 (PC2) is a Ca(2+)-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca(2+). PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca(2+)-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca(2+) remains unknown. We propose that the PC2 EF-hand is a Ca(2+) sensor required for channel gating. Consistent with this, Ca(2+) binding causes a dramatic decrease in the radius of gyration (R(g)) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca(2+) concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D(max)) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca(2+) via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca(2+) release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca(2+) levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca(2+)-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.

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.

In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca²⁺) by isothermal titration calorimetry and undergo Ca²⁺-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca²⁺ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca²⁺-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease.

New A-ring pyridine fused androstanes in 17a-homo-17-oxa (D-homo lactone), 17α-picolyl or 17(E)-picolinylidene series were synthesized and validated by X-ray crystallography, HRMS, IR and NMR spectroscopy. Novel compounds 3, 5, 8 and 12 were prepared by treatment of 4-en-3-one or 4-ene-3,6-dione D-modified androstane derivatives with propargylamine catalyzed by Cu(II), and evaluated for potential anticancer activity in vitro using human cancer cell lines and recombinant targets of steroidal anti-cancer drugs. Pyridine fusion to position 3,4 of the A-ring may dramatically enhance affinity of 17α-picolyl compounds for CYP17 while conferring selective antiproliferative activity against PC-3 cells. Similarly, pyridine fusion to the A-ring of steroidal D-homo lactones led to identification of new inhibitors of aldo–keto reductase 1C3, an enzyme targeted in acute myeloid leukemia, breast and prostate cancers. One A-pyridine D-lactone steroid 5 also has selective submicromolar antiproliferative activity against HT-29 colon cancer cells. None of the new derivatives have affinity for estrogen or androgen receptors in a yeast screen, suggesting negligible estrogenicity and androgenicity. Combined, our results suggest that A-ring pyridine fusions have potential in modulating the anticancer activity of steroidal compounds.