A Protein Interaction Network for Ecm29 Links the 26 S Proteasome to Molecular Motors and Endosomal Components

Ecm29 is a 200-kDa HEAT repeat protein that binds the 26 S proteasome. Genome-wide two-hybrid screens and mass spectrometry have identified molecular motors, endosomal components, and ubiquitin-proteasome factors as Ecm29-interacting proteins. The C-terminal half of human Ecm29 binds myosins and kinesins; its N-terminal region binds the endocytic proteins, Vps11, Rab11-FIP4, and rabaptin. Whereas full-length FLAG-Ecm29, its C-terminal half, and a small central fragment of Ecm29 remain bound to glycerol-gradient-separated 26 S proteasomes, the N-terminal half of Ecm29 does not. Confocal microscopy showed that Ecm-26 S proteasomes are present on flotillin-positive endosomes, but they are virtually absent from caveolin- and clathrin-decorated endosomes. Expression of the small central fragment of Ecm29 markedly reduces proteasome association with flotillin-positive endosomes. Identification of regions within Ecm29 capable of binding molecular motors, endosomal proteins, and the 26 S proteasome supports the hypothesis that Ecm29 serves as an adaptor for coupling 26 S proteasomes to specific cellular compartments.     

Characterization of mammalian Ecm29, a 26 S proteasome-associated protein that localizes to the nucleus and membrane vesicles

In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.     

Comparison of ARM and HEAT protein repeats

ARM and HEAT motifs are tandemly repeated sequences of approximately 50 amino acid residues that occur in a wide variety of eukaryotic proteins. An exhaustive search of sequence databases detected new family members and revealed that at least 1 in 500 eukaryotic protein sequences contain such repeats. It also rendered the similarity between ARM and HEAT repeats, believed to be evolutionarily related, readily apparent. All the proteins identified in the database searches could be clustered by sequence similarity into four groups: canonical ARM-repeat proteins and three groups of the more divergent HEAT-repeat proteins. This allowed us to build improved sequence profiles for the automatic detection of repeat motifs. Inspection of these profiles indicated that the individual repeat motifs of all four classes share a common set of seven highly conserved hydrophobic residues, which in proteins of known three-dimensional structure are buried within or between repeats. However, the motifs differ at several specific residue positions, suggesting important structural or functional differences among the classes. Our results illustrate that ARM and HEAT-repeat proteins, while having a common phylogenetic origin, have since diverged significantly. We discuss evolutionary scenarios that could account for the great diversity of repeats observed.     

Topological characteristics of helical repeat proteins.

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.     

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.     

ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.     

Identification and analysis of novel tandem repeats in the cell surface proteins of archaeal and bacterial genomes using computational tools

We have identified four novel repeats and two domains in cell surface proteins encoded by the Methanosarcina acetivorans genome and in some archaeal and bacterial genomes. The repeats correspond to a certain number of amino acid residues present in tandem in a protein sequence and each repeat is characterized by conserved sequence motifs. These correspond to: (a) a 42 amino acid (aa) residue RIVW repeat; (b) a 45 aa residue LGxL repeat; (c) a 42 aa residue LVIVD repeat; and (d) a 54 aa residue LGFP repeat. The domains correspond to a certain number of aa residues in a protein sequence that do not comprise internal repeats. These correspond to: (a) a 200 aa residue DNRLRE domain; and (b) a 70 aa residue PEGA domain. We discuss the occurrence of these repeats and domains in the different proteins and genomes analysed in this work.     

The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport

Importin-beta is a nuclear transport factor which mediates the nuclear import of various nuclear proteins. The N-terminal 1-449 residue fragment of mouse importin-beta (impbeta449) possesses the ability to bidirectionally translocate through the nuclear pore complex (NPC), and to bind RanGTP. The structure of the uncomplexed form of impbeta449 has been solved at a 2.6 A resolution by X-ray crystallography. It consists of ten copies of the tandemly arrayed HEAT repeat and exhibits conformational flexibility which is involved in protein-protein interaction for nuclear transport. The overall conformation of the HEAT repeats shows that a twisted motion produces a significantly varied superhelical architecture from the previously reported structure of RanGTP-bound importin-beta. These conformational changes appear to be the sum of small conformational changes throughout the polypeptide. Such a flexibility, which resides in the stacked HEAT repeats, is essential for interaction with RanGTP or with NPCs. Furthermore, it was found that impbeta449 has a structural similarity with another nuclear migrating protein, namely beta-catenin, which is composed of another type of helix-repeated structure of ARM repeat. Interestingly, the essential regions for NPC translocation for both importin-beta and beta-catenin are spatially well overlapped with one another. This strongly indicates the importance of helix stacking of the HEAT or ARM repeats for NPC-passage.     

The role of introns in repeat protein gene formation

Genes composed of tandem repetitive sequence motifs are abundant in nature and are enriched in eukaryotes. To investigate repeat protein gene formation mechanisms, we have conducted a large-scale analysis of their introns and exons. We find that a wide variety of repeat motifs exhibit a striking conservation of intron position and phase, and are composed of exons that encode one or two complete repeats. These results suggest a simple model of repeat protein gene formation from local duplications. This model is corroborated by amino acid sequence similarity patterns among neighboring repeats from various repeat protein genes. The distribution of one- and two-repeat exons indicates that intron-facilitated repeat motif duplication, in which the start and end points of duplication are located in consecutive intronic regions, significantly exceeds intron-independent duplication. These results suggest that introns have contributed to the greater abundance of repeat protein genes in eukaryotic versus prokaryotic organisms, a conclusion that is supported by taxonomic analysis.     

Proteasome activator 200: the heat is on..

Proteasomes play a key regulatory role in all eukaryotic cells by removing proteins in a timely manner. There are two predominant forms: The 20S core particle (CP) can hydrolyze peptides and certain unstructured proteins, and the 26S holoenzyme is able to proteolyse most proteins conjugated to ubiquitin. The 26S complex consists of a CP barrel with a 19S regulatory particle (RP; a.k.a PA700) attached to its outer surface. Several studies purified another proteasome activator with a MW of 200 kDa (PA200) that attaches to the same outer ring of the CP. A role for PA200 has been demonstrated in spermatogenesis, in response to DNA repair and in maintenance of mitochondrial inheritance. Enhanced levels of PA200-CP complexes are observed under conditions in which either activated or disrupted CP prevail, suggesting it participates in regulating overall proteolytic activity. PA200, or its yeast ortholog Blm10, may also incorporate into 26S proteasomes yielding PA200-CP-RP hybrids. A three-dimensional molecular structure determined by x-ray crystallography of Blm10-CP provides a model for activation. The carboxy terminus of Blm10 inserts into a dedicated pocket in the outer ring of the CP surface, whereas multiple HEAT-like repeats fold into an asymmetric solenoid wrapping around the central pore to stabilize a partially open conformation. The resulting hollow domelike structure caps the entire CP surface. This asymmetric structure may provide insight as to how the 19S RP, with two HEAT repeatlike subunits (Rpn1, Rpn2) alongside six ATPases (Rpt1-6), attaches to the same surface of the CP ring, and likewise, induces pore opening.     

Electrostatic interactions involving the extreme C terminus of nuclear export factor CRM1 modulate its affinity for cargo

The toroid-shaped nuclear protein export factor CRM1 is constructed from 21 tandem HEAT repeats, each of which contains an inner (B) and outer (A) α-helix joined by loops. Proteins targeted for export have a nuclear export signal (NES) that binds between the A-helices of HEAT repeats 11 and 12 on the outer surface of CRM1. RanGTP binding increases the affinity of CRM1 for NESs. In the absence of RanGTP, the CRM1 C-terminal helix, together with the HEAT repeat 9 loop, modulates its affinity for NESs. Here we show that there is an electrostatic interaction between acidic residues at the extreme distal tip of the C-terminal helix and basic residues on the HEAT repeat 12 B-helix that lies on the inner surface of CRM1 beneath the NES binding site. Small angle x-ray scattering indicates that the increased affinity for NESs generated by mutations in the C-terminal helix is not associated with large scale changes in CRM1 conformation, consistent with the modulation of NES affinity being mediated by a local change in CRM1 near the NES binding site. These data also suggest that in the absence of RanGTP, the C-terminal helix lies across the CRM1 toroid in a position similar to that seen in the CRM1-Snurportin crystal structure. By creating local changes that stabilize the NES binding site in its closed conformation and thereby reducing the affinity of CRM1 for NESs, the C-terminal helix and HEAT 9 loop facilitate release of NES-containing cargo in the cytoplasm and also inhibit their return to the nucleus.     

Aromatic di-alanine repeats (AdAR) are structural motifs characteristic of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) family

The aromatic di-alanine repeat is a novel 12-amino acid-long motif constituting alternate small and large hydrophobic residues that mediate the close packing of alpha-helices. A hidden Markov model profile was constructed from the motifs initially described in Soluble N-ethyl maleimide-sensitive factor attachment proteins (SNAP), a family of soluble proteins involved in intracellular membrane fusion. Scanning different sets of protein sequences showed unambiguously that this profile defines a structural motif independent of the tetratrico peptide repeat, another widespread alpha-helical motif. In addition to SNAP, aromatic di-alanine repeats are found in selective LIM homeodomain binding proteins (SLB) and in proteins from the Pyrococcus and Archaeoglobus prokaryotes.     

Helical repeat structure of apoptosis inhibitor 5 reveals protein-protein interaction modules

Apoptosis inhibitor 5 (API5) is an anti-apoptotic protein that is up-regulated in various cancer cells. Here, we present the crystal structure of human API5. API5 exhibits an elongated all α-helical structure. The N-terminal half of API5 is similar to the HEAT repeat and the C-terminal half is similar to the ARM (Armadillo-like) repeat. HEAT and ARM repeats have been implicated in protein-protein interactions, suggesting that the cellular roles of API5 may be to mediate protein-protein interactions. Various components of multiprotein complexes have been identified as API5-interacting protein partners, suggesting that API5 may act as a scaffold for multiprotein complexes. API5 exists as a monomer, and the functionally important heptad leucine repeat does not exhibit the predicted a dimeric leucine zipper. Additionally, Lys-251, which can be acetylated in cells, plays important roles in the inhibition of apoptosis under serum deprivation conditions. The acetylation of this lysine also affects the stability of API5 in cells.     

Karyopherin flexibility in nucleocytoplasmic transport

Recent structural work on nuclear transport factors of the importin-beta superfamily of karyopherins has shown that these proteins are superhelices of HEAT repeats that are able to assume different conformations in different functional states. The inherent flexibility of these helicoids facilitates the accommodation of different binding partners by an induced-fit type of mechanism. Moreover, the energy stored by distorting these molecules may partially balance binding energies to enable assembly and disassembly of their complexes with relatively small energy changes. Flexibility appears to be an intrinsic feature of such superhelices and might be functionally important not only for karyopherins and nuclear transport, but also for HEAT repeat proteins from other biological systems.     

Evolution of the tandem repeats in thymidylate synthase enhancer region (TSER) in primates

The upstream regulatory region of the human thymidylate synthase gene (thymidylate synthase enhancer region, TSER) is length polymorphic, attributable to variable numbers of tandemly repeated copies of a 28-bp fragment. It has been found that TSER length polymorphism is correlated to malignancy risk. To further our understanding of the origin and evolution of TSER, this region was investigated among different primates, including hominoids, two subfamilies of the Old World monkeys (OWMs): colobines and cercopithecines, and two species of the New World monkeys (NWMs). In addition to humans, our results show that length polymorphism in TSER is also present in some primate populations, although it appears that this region is length monomorphic in many other primates. We identified three unique repeat motifs in TSER and defined them as R1, R2, and R3, respectively, starting from the 3' end. The same repeat motifs from different species are more similar to each other than different repeat motifs within same species are. Such a paraphyletic pattern suggests that divergence of the three repeat motifs predated divergence of the OWMs/hominoids and the NWMs. The most recent common ancestor (MRCA) of hominoids and the OWMs probably possessed triple repeats but now double and triple repeats are two dominant types in hominoids and the OWMs. In addition, our results show that each of the three repeat motifs may be lost independently. We have also found clues that recombination was involved in formation of tandem repeat polymorphism in TSER.     

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5′TOP sequence

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5′TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5′ UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5′TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.     

Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.     

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.