Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30−34 amino acid residues and exclusively functions to mediate protein−protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix−turn−helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix−turn−helix bundles with relatively flexible loops. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. Recent studies have demonstrated that ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its partner. In addition, the availability of thousands of ankyrin repeat sequences has made it feasible to use rational design to modify the specificity and stability of physiologically important ankyrin repeat proteins and even to generate ankyrin repeat proteins with novel functions through combinatorial chemistry approaches.

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The solution structure of the tumor suppressor p16INK4A has been determined by NMR, and important recognition regions of both cdk4 and p16INK4A have been identified. The tertiary structure of p16INK4A contains four helix-turn-helix motifs linked by three loops. Twelve tumorigenic mutants of p16INK4A have been constructed and analyzed for their structure and activity, and new mutants have been designed rationally. A fragment of 58 residues at the N terminus of cdk4 important for p16INK4A binding has been identified. The importance of this region was further verified by mutational analysis of cdk4. These results and docking experiments have been used to assess possible modes of binding between p16INK4A and cdk4.

P16INK4A (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly due to the prevalence of genetic inactivation of the p16INK4A (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that elevated expression (up-regulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications in the structure-function relationship of P16 and in human cancers.

Abstract-Endothelial nitric oxide synthase (eNOS) plays a central role in maintaining cardiovascular homeostasis by controlling NO bioavailability. The activity of eNOS in vascular endothelial cells (ECs) largely depends on posttranslational modifications, including phosphorylation. Because the activity of AMP-activated protein kinase (AMPK) in ECs can be increased by multiple cardiovascular events, we studied the phosphorylation of eNOS Ser633 by AMPK and examined its functional relevance in the mouse models. Shear stress, atorvastatin, and adiponectin all increased AMPK Thr172 and eNOS Ser633 phosphorylations, which were abolished if AMPK was pharmacologically inhibited or genetically ablated. The constitutively active form of AMPK or an AMPK agonist caused a sustained Ser633 phosphorylation. Expression of gain-/loss-of-function eNOS mutants revealed that Ser633 phosphorylation is important for NO production. The aorta of AMPK␣2 Ϫ/Ϫ mice showed attenuated atorvastatin-induced eNOS phosphorylation. Nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) confirmed that eNOS Ser633 was able to compete with Ser1177 or acetyl-coenzyme A carboxylase Ser79 for AMPK␣ phosphorylation. Nano-LC/MS/MS confirmed that eNOS purified from AICAR-treated ECs was phosphorylated at both Ser633 and Ser1177. Our results indicate that AMPK phosphorylation of eNOS Ser633 is a functional signaling event for NO bioavailability in ECs.

DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.

The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in metal ion catalysis of the chemical step. Here we report a functional intermediate structure of a DNA polymerase with only one metal ion bound, the DNA polymerase beta-DNA template-primer-chromium(III).2'-deoxythymidine 5'-beta,gamma-methylenetriphosphate [Cr(III).dTMPPCP] complex, at 2.6 A resolution. The complex is distinct from the structures of other polymerase-DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydroxyl group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnover in biochemical assays of crystallized protein as well as from the determination that soaking Pol beta crystals with Mn(II) ions leads to formation of the product complex, Pol beta-DNA-Cr(III).PCP, whose structure is also reported. An important feature of both structures is that the fingers subdomain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of the fingers subdomain is induced specifically by binding of the metal-dNTP complex prior to binding of the catalytic Mg(2+) ion. This has led us to reevaluate our previous evidence regarding the existence of a rate-limiting conformational change in Pol beta's reaction pathway. The results of stopped-flow studies suggest that there is no detectable rate-limiting conformational change step.

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.