Crystal Structure of <Emphasis Type="Italic">Human</Emphasis> ASB9-2 and Substrate-Recognition of CKB

Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9) is a specific substrate-recognition subunit of an elongin C-cullin-SOCS box E3 ubiquitin ligase complex. It recognizes its substrate, brain type creatine kinase (CKB), using the ankyrin repeat domain; and facilitates the polyubiquitination of CKB to mediate proteasomal degradation through the SOCS box domain. HASB9-2 is an isoform of hASB9 that contains one ankyrin repeat domain. In this study, the crystal structure of hASB9-2 is shown at 2.2-Å resolution using molecular replacement. Overall, hASB9-2 forms a slightly curved arch with a characteristic L-shaped cross-section. Amino acid substitution analysis based on docking experiments revealed that His103 and Phe107 in hASB9-2 are essential for binding to CKB. Analysis of truncation mutants demonstrated that the first six ankyrin repeats along with the N-terminal region of hASB9-2 contribute to the interaction with CKB.     

ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function

Background  

The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9).     

The SOCS box-adapting proteins for ubiquitination and proteasomal degradation

The suppressor of cytokine signalling (SOCS) box was first identified in the SH2-containing SOCS box family (cytokine-inducible SH2-containing protein, SOCS1-7) and is a 40-amino acid motif, which functions to recruit an E3 ubiquitin ligase complex consisting of the adapter proteins elongins B and C, Rbx2 and the scaffold protein Cullin5. The SOCS box is found in a diverse array of intracellular signalling molecules, many of which contain different protein interaction domains such as SPRY and WD40 domains, leucine and ankyrin repeats or other functional domains such as GTPases. In general, the SOCS box-containing proteins are thought to act as substrate-recognition modules to mediate the polyubiquitination and subsequent degradation of substrate proteins by the 26S proteasome.     

Ankyrin repeat and suppressors of cytokine signaling box protein asb-9 targets creatine kinase B for degradation

The suppressors of cytokine signaling (SOCS) proteins inhibit cytokine action by direct interaction with Janus kinases or activated cytokine receptors. In addition to the N-terminal and Src homology 2 domains that mediate these interactions, SOCS proteins contain a C-terminal SOCS box. DNA data base searches have identified a number of other protein families that possess a SOCS box, of which the ankyrin repeat and SOCS box-containing (Asb) proteins constitute the largest. Although it is known that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological and biochemical functions of the Asbs are largely undefined. Using a proteomics approach, we demonstrate that creatine kinase B (CKB) interacts with Asb-9 in a specific, SOCS box-independent manner. This interaction increases the polyubiquitylation of CKB and decreases total CKB levels within the cell. The targeting of CKB for degradation by Asb-9 was primarily SOCS box-dependent and suggests that Asb-9 acts as a specific ubiquitin ligase regulating levels of this evolutionarily conserved enzyme.     

Structural basis for recognition of 2',5'-linked oligoadenylates by human ribonuclease L

An interferon-induced endoribonuclease, ribonuclease L (RNase L), is implicated in both the molecular mechanism of action of interferon and the fundamental control of RNA stability in mammalian cells. RNase L is catalytically active only after binding to an unusual activator molecule containing a 5'-phosphorylated 2',5'-linked oligoadenylate (2-5A), in the N-terminal half. Here, we report the crystal structure of the N-terminal ankyrin repeat domain (ANK) of human RNase L complexed with the activator 2-5A. This is the first structural view of an ankyrin repeat structure directly interacting with a nucleic acid, rather than with a protein. The ANK domain folds into eight ankyrin repeat elements and forms an extended curved structure with a concave surface. The 2-5A molecule is accommodated at a concave site and directly interacts with ankyrin repeats 2-4. Interestingly, two structurally equivalent 2-5A binding motifs are found at repeats 2 and 4. The structural basis for 2-5A recognition by ANK is essential for designing stable 2-5As with a high likelihood of activating RNase L.     

Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle

Ankyrin repeat and SOCS box protein 15 (ASB15) is an Asb family member expressed predominantly in skeletal muscle. We have previously reported that ASB15 mRNA abundance decreases after administration of beta-adrenergic receptor agonists. Because beta-adrenergic receptor agonists are known to stimulate muscle hypertrophy, the objective of this study was to determine whether ASB15 regulates cellular processes that contribute to muscle growth. Stable myoblast C2C12 cells expressing full-length ASB15 (ASB15-FL) and ASB15 lacking the ankyrin repeat (ASB15-Ank) or SOCS box (ASB15-SOCS) motifs were evaluated for changes in proliferation, differentiation, protein synthesis, and protein degradation. Expression of ASB15-FL caused a delay in differentiation, followed by an increase in protein synthesis of approximately 34% (P<0.05). A consistent effect of ASB15 overexpression was observed in vivo, where ectopic expression of ASB15 increased skeletal muscle fiber area (P<0.0001) after 9 days. Expression of ASB15-SOCS altered differentiation of myoblasts, resulting in detachment of cells from culture plates. Expression of ASB15-Ank increased protein degradation by 84 h of differentiation (P<0.05), and in vivo ectopic expression of an ASB15 construct lacking both the ankyrin repeat and SOCS box motifs decreased skeletal muscle fiber area (P<0.0001). Together, these results suggest ASB15 participates in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. This is the first study to demonstrate a role for an Asb family member in skeletal muscle growth.     

Structural insights for MPP8 chromodomain interaction with histone H3 lysine 9: potential effect of phosphorylation on methyl-lysine binding

M-phase phosphoprotein 8 (MPP8) harbors an N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/H3K9me2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine-9-containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80 and Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side-chain hydroxyl oxygen of Tyr83 suggests that the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).     

稳定表达人ASB12的C2C12细胞系的建立及鉴定

ASB12(homo sapiens ankyrin repeat and SOCS box containing 12)蛋白含有5个ANK(ankyrin repeat sequence)序列和一个保守的SOCS(suppressor of cytokine signaling)盒结构域,是ASBs(human ankyrin repeat andSOCS box containing protein family,ASB family)家族的成员.人类ASB12基因在成体心肌和骨骼肌组织中特异表达,是成肌分化的候选基因.利用阳离子聚合物转染技术将重组表达质粒pCMV-tag2B-ASB12转染小鼠骨骼肌细胞系C2C12细胞,通过G418筛选、免疫荧光检测、RT-PCR分析、Western blotting检测建立了稳定表达ASB12的细胞系C2C12-ASB12,为研究ASB12在骨骼肌发育及其相关功能提供有用的细胞研究模型.     

Ankyrin repeat: a unique motif mediating protein-protein interactions

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.     

Structural and functional analysis of the U3 snoRNA binding protein Rrp9

The U3 snoRNA is required for 18S rRNA processing and small subunit ribosome formation in eukaryotes. Different from other box C/D snoRNAs, U3 contains an extra 5′ domain that pairs with pre-rRNA and a unique B/C motif essential for recruitment of the U3-specific Rrp9 protein. Here, we analyze the structure and function of Rrp9 with crystallographic, biochemical, and cellular approaches. Rrp9 is composed of a WD repeat domain and an N-terminal region. The crystal structures of the WD domain of yeast Rrp9 and its human ortholog U3-55K were determined, revealing a typical seven-bladed propeller fold. Several conserved surface patches on the WD domain were identified, and their function in RNP assembly and yeast growth were analyzed by mutagenesis. Prior association of Snu13 with the B/C motif was found to enhance the specific binding of the WD domain. We show that a conserved 7bc loop is crucial for specific recognition of U3, nucleolar localization of Rrp9, and yeast growth. In addition, we show that the N-terminal region of Rrp9 contains a bipartite nuclear localization signal that is dispensable for nucleolar localization. Our results provide insight into the functional sites of Rrp9.     

Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins

The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.     

Structure and stability of the ankyrin domain of the Drosophila Notch receptor

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.     

心脏特异表达基因ASBIO的克隆及表达

从人类心脏文库中成功克隆了一个新的含有锚蛋白重复序列及SOCS盒结构域蛋白（ Ankyrin repeat and SOCS box protein, ASB）的家族成员ASBIO。该基因定位于染色体7q36．1,开放阅读框包含1329个核苷酸,由7个锚蛋白重复序列和1个SOCS盒组成,编码452个氨基酸,分子量大约是49．5kD。物种间的进化型分析比较表明该基因是非常保守的,人的ASB10基因和小鼠的Asb10基因有84．8％的同源性。半定量RT-PCR实验结果证明Asb10基因在成年小鼠的心脏和肌肉组织中高表达,提示其可能与心脏和肌肉组织的发育有关。亚细胞定位显示ASB10蛋白在细胞质和细胞核均有表达,为进一步研究奠定了基础。     

An ankyrin repeat protein is involved in anthocyanin biosynthesis in Arabidopsis

The ankyrin domain is one of the most common protein motifs in eukaryotic proteins. Repeated ankyrin domains are ubiquitous and their mediation of protein-protein interactions is involved in a number of physiological and developmental responses such as the cell cycle, signal transduction and cell differentiation. A novel putative phytochrome-interacting ankyrin repeat protein 2 (PIA2) containing three repeated ankyrin domains was identified in Arabidopsis. An in vitro pull-down and phosphorylation assay revealed that PIA2 is phosphorylated and interacts directly with oat phytochrome A. The N-terminal domain of PIA2 was specifically phosphorylated, whereas interactions between the domains of PIA2 and phytochrome A had no Pr/Pfr preference. PIA2 was ubiquitously expressed in most tissues and was localized in both the nucleus and the cytoplasm independent of treatment with light of specific wavelengths. Anthocyanin accumulation in seedlings grown under far-red light, a typical phenotype of wild-type plants, was reduced in a loss-of-function mutant of PIA2 (pia2), whereas anthocyanin accumulation was increased in an overexpressing plant (PIA2-OX). The gene expression of UDP-flavonoid-3'-glucosyl-transferase (UF3GT), a major enzyme in the anthocyanin biosynthesis processes, was decreased in pia2 knockout plants suggesting that decreased anthocyanin was because of the decreased expression of UF3GT. Our results suggest that PIA2 plays a role in the anthocyanin biosynthesis during seedling development as a novel phytochrome-interacting protein.     

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies.     

Functional characterization of 2',5'-linked oligoadenylate binding determinant of human RNase L

RNase L is activated by the binding of unusual 2',5'-linked oligoadenylates (2-5A) and acts as the effector enzyme of the 2-5A system, an interferon-induced anti-virus mechanism. Efforts have been made to understand the 2-5A binding mechanism, not only for scientific interests but also for the prospects that the understanding of such mechanisms lead to new remedies for viral diseases. We have recently elucidated the crystal structure of the 2-5A binding ankyrin repeat domain of human RNase L complexed with 2-5A. To determine the contributions of amino acid residues surrounding the 2-5A binding site, point mutants and a deletion mutant were designed based on the crystal structure. These mutant proteins were analyzed for their interaction with 2-5A using a steady-state fluorescence technique. In addition, full-length RNase L mutants were tested for their activation by 2-5A. The results reveal that pi-pi stacking interactions of Trp60 and Phe126, electrostatic interactions of Lys89 and Arg155, and hydrogen bonding by Glu131 make crucial contributions to 2-5A binding. It was also found that the crystal structure of the ankyrin repeat domain L.2-5A complex accurately portrays the 2-5A binding mode in full-length RNase L.     

High-affinity binders selected from designed ankyrin repeat protein libraries

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.     

Crystal structure of the human TRPV2 channel ankyrin repeat domain

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.