The Four Hydrophobic Residues on the Hsp70 Inter-Domain Linker Have Two Distinct Roles

The ubiquitous molecular chaperone 70-kDa heat shock proteins (Hsp70) play key roles in maintaining protein homeostasis. Hsp70s contain two functional domains: a nucleotide binding domain and a substrate binding domain. The two domains are connected by a highly conserved inter-domain linker, and allosteric coupling between the two domains is critical for chaperone function. The auxiliary chaperone 40-kDa heat shock proteins (Hsp40) facilitate all the biological processes associated with Hsp70s by stimulating the ATPase activity of Hsp70s. Although an overall essential role of the inter-domain linker in both allosteric coupling and Hsp40 interaction has been suggested, the molecular mechanisms remain largely unknown. Previously, we reported a crystal structure of a full-length Hsp70 homolog, in which the inter-domain linker forms a well-ordered β strand. Four highly conserved hydrophobic residues reside on the inter-domain linker. In DnaK, a well-studied Hsp70, these residues are V389, L390, L391, and L392. In this study, we biochemically dissected their roles. The inward-facing side chains of V389 and L391 form extensive hydrophobic contacts with the nucleotide binding domain, suggesting their essential roles in coupling the two functional domains, a hypothesis confirmed by mutational analysis. On the other hand, L390 and L392 face outward on the surface. Mutation of either abolishes DnaK's in vivo function, yet intrinsic biochemical properties remain largely intact. In contrast, Hsp40 interaction is severely compromised. Thus, for the first time, we separated the two essential roles of the highly conserved Hsp70 inter-domain linker: coupling the two functional domains through V389 and L391 and mediating the interaction with Hsp40 through L390 and L392.     

Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor

The BRCT (BRCA1 C-terminus) is an evolutionary conserved protein-protein interacting module found as single, tandem or multiple repeats in a diverse range of proteins known to play roles in the DNA-damage response. The BRCT domains of 53BP1 bind to the tumour suppressor p53. To investigate the nature of this interaction, we have determined the crystal structure of the 53BP1 BRCT tandem repeat in complex with the DNA-binding domain of p53. The structure of the 53BP1-p53 complex shows that the BRCT tandem repeats pack together through a conserved interface that also involves the inter-domain linker. A comparison of the structure of the BRCT region of 53BP1 with the BRCA1 BRCT tandem repeat reveals that the interdomain interface and linker regions are remarkably well conserved. 53BP1 binds to p53 through contacts with the N-terminal BRCT repeat and the inter-BRCT linker. The p53 residues involved in this binding are mutated in cancer and are also important for DNA binding. We propose that BRCT domains bind to cellular target proteins through a conserved structural element termed the 'BRCT recognition motif'.     

Model for the three-dimensional structure of vitronectin: predictions for the multi-domain protein from threading and docking.

The structure of vitronectin, an adhesive protein that circulates in high concentrations in human plasma, was predicted through a combination of computational methods and experimental approaches. Fold recognition and sequence-structure alignment were performed using the threading program PROSPECT for each of three structural domains, i.e., the N-terminal somatomedin B domain (residues 1-53), the central region that folds into a four-bladed beta-propeller domain (residues 131-342), and the C-terminal heparin-binding domain (residues 347-459). The atomic structure of each domain was generated using MODELLER, based on the alignment obtained from threading. Docking experiments between the central and C-terminal domains were conducted using the program GRAMM, with limits on the degrees of freedom from a known inter-domain disulfide bridge. The docked structure has a large inter-domain contact surface and defines a putative heparin-binding groove at the inter-domain interface. We also docked heparin together with the combined structure of the central and C-terminal domains, using GRAMM. The predictions from the threading and docking experiments are consistent with experimental data on purified plasma vitronectin pertaining to protease sensitivity, ligand-binding sites, and buried cysteines.     

Analysis of the quaternary structure of the MutL C-terminal domain

The dimeric DNA mismatch repair protein MutL has a key function in communicating mismatch recognition by MutS to downstream repair processes. Dimerization of MutL is mediated by the C-terminal domain, while activity of the protein is modulated by the ATP-dependent dimerization of the highly conserved N-terminal domain. Recently, a crystal structure analysis of the Escherichia coli MutL C-terminal dimerization domain has been reported and a model for the biological dimer was proposed. In this model, dimerization is mediated by the internal (In) subdomain comprising residues 475-569. Here, we report a computational analysis of all protein interfaces observed in the crystal structure and suggest that the biological dimer interface is formed by a hydrophobic surface patch of the external (Ex) subdomain (residues 432-474 and 570-615). Moreover, sequence analysis revealed that this surface patch is conserved among the MutL proteins. To test this hypothesis, single and double-cysteine variants of MutL were generated and tested for their ability to be cross-linked with chemical cross-linkers of various size. Finally, deletion of the C-terminal residues 605-615 abolished homodimerization. The biochemical data are fully compatible with a revised model for the biological dimer, which has important implications for understanding the heterodimerization of eukaryotic MutL homologues, modeling the MutL holoenzyme and predicting protein-protein interaction sites.     

Structure of the MID1 tandem B-boxes reveals an interaction reminiscent of intermolecular ring heterodimers

The tripartite motif (TRIM) protein family, defined by N-terminal RING, B-box, and coiled-coil (RBCC) domains, consists of either a single type 2 B-box domain or tandem B-box domains of type 1 and type 2 (B1B2). Here, we report the first structure of the B-box domains in their native tandem orientation. The B-boxes are from Midline-1, a putative ubiquitin E3 ligase that is required for the proteosomal degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). This function of MID1 is facilitated by the direct binding of Alpha4, a regulatory subunit of PP2Ac, to B-box1, while B-box2 appears to influence this interaction. Both B-box1 and B-box2 bind two zinc atoms in a cross-brace motif and adopt a similar betabetaalpha structure reminiscent of the RING, PHD, ZZ, and U-box domains, although they differ from each other and with RING domains in the spacing of their zinc-binding residues. The two B-box domains pack against each other with the interface formed by residues located on the structured loop consisting of the two antiparallel beta-strands. The surface area of the interface is 188 A2 (17% of the total surface). Consistent with the globular structure, the Tm of the tandem B-box domain (59 degrees C) is higher than the individual domains, supporting a stable interaction between the B-box 1 and 2 domains. Notably, the interaction is reminiscent of the interaction of recently determined RING dimers, suggesting the possibility of an evolutionarily conserved role for B-box2 domains in regulating functional RING-type folds.     

Crystal structures of the conserved tRNA-modifying enzyme GidA: implications for its interaction with MnmE and substrate

GidA is a flavin-adenine-dinucleotide (FAD)-binding protein that is conserved among bacteria and eucarya. Together with MnmE, it is involved in the addition of a carboxymethylaminomethyl group to the uridine base in the wobble position (nucleotide 34) of tRNAs that read split codon boxes. Here, we report the crystal structures of the GidA proteins from both Escherichia coli and Chlorobium tepidum. The structures show that the protein can be divided into three domains: a first FAD-binding domain showing the classical Rossmann fold, a second α/β domain inserted between two strands of the Rossmann fold, and an α-helical C-terminal domain. The domain inserted into the Rossmann fold displays structural similarity to the nicotinamide-adenine-dinucleotide-(phosphate)-binding domains of phenol hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, and, correspondingly, we show that GidA binds NADH with high specificity as an initial donor of electrons. GidA behaves as a homodimer in solution. As revealed by the crystal structures, homodimerization is mediated via both the FAD-binding domain and the NADH-binding domain. Finally, a large patch of highly conserved, positively charged residues on the surface of GidA leading to the FAD-binding site suggests a tRNA-binding surface. We propose a model for the interaction between GidA and MnmE, which is supported by site-directed mutagenesis. Our data suggest that this interaction is modulated and potentially regulated by the switch function of the G domain of MnmE.     

Folding of beta/alpha-unit scrambled forms of S. cerevisiae triosephosphate isomerase: Evidence for autonomy of substructure formation and plasticity of hydrophobic and hydrogen bonding interactions in core of (beta/alpha)8-barrel

We show that residues at the interfaces of protein-protein complexes have higher side-chain energy than other surface residues. Eight different sets of protein complexes were analyzed. For each protein pair, the complex structure was used to identify the interface residues in the unbound monomer structures. Side-chain energy was calculated for each surface residue in the unbound monomer using our previously developed scoring function.1 The mean energy was calculated for the interface residues and the other surface residues. In 15 of the 16 monomers, the mean energy of the interface residues was higher than that of other surface residues. By decomposing the scoring function, we found that the energy term of the buried surface area of non-hydrogen-bonded hydrophilic atoms is the most important factor contributing to the high energy of the interface regions. In spite of lacking hydrophilic residues, the interface regions were found to be rich in buried non-hydrogen-bonded hydrophilic atoms. Although the calculation results could be affected by the inaccuracy of the scoring function, patch analysis of side-chain energy on the surface of an isolated protein may be helpful in identifying the possible protein-protein interface. A patch was defined as 20 residues surrounding the central residue on the protein surface, and patch energy was calculated as the mean value of the side-chain energy of all residues in the patch. In 12 of the studied monomers, the patch with the highest energy overlaps with the observed interface. The results are more remarkable when only three residues with the highest energy in a patch are averaged to derive the patch energy. All three highest-energy residues of the top energy patch belong to interfacial residues in four of the eight small protomers. We also found that the residue with the highest energy score on the surface of a small protomer is very possibly the key interaction residue.     

A conserved membrane-spanning amino acid motif drives homomeric and supports heteromeric assembly of presynaptic SNARE proteins

Assembly of the SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 to binary and ternary complexes is important for docking and/or fusion of presynaptic vesicles to the neuronal plasma membrane prior to regulated neurotransmitter release. Despite the well characterized structure of their cytoplasmic assembly domains, little is known about the role of the transmembrane segments in SNARE protein assembly and function. Here, we identified conserved amino acid motifs within the transmembrane segments that are required for homodimerization of synaptobrevin II and syntaxin 1A. Minimal motifs of 6-8 residues grafted onto an otherwise monomeric oligoalanine host sequence were sufficient for self-interaction of both transmembrane segments in detergent solution or membranes. These motifs constitute contiguous areas of interfacial residues assuming alpha-helical secondary structures. Since the motifs are conserved, they also contributed to heterodimerization of synaptobrevin II and syntaxin 1A and therefore appear to constitute interaction domains independent of the cytoplasmic coiled coil regions. Interactions between the transmembrane segments may stabilize the SNARE complex, cause its multimerization to previously observed multimeric superstructures, and/or be required for the fusogenic activity of SNARE proteins.     

Structure of the VHS domain of human Tom1 (target of myb 1): insights into interactions with proteins and membranes

VHS domains are found at the N-termini of select proteins involved in intracellular membrane trafficking. We have determined the crystal structure of the VHS domain of the human Tom1 (target of myb 1) protein to 1.5 A resolution. The domain consists of eight helices arranged in a superhelix. The surface of the domain has two main features: (1) a basic patch on one side due to several conserved positively charged residues on helix 3 and (2) a negatively charged ridge on the opposite side, formed by residues on helix 2. We compare our structure to the recently obtained structure of tandem VHS-FYVE domains from Hrs [Mao, Y., Nickitenko, A., Duan, X., Lloyd, T. E., Wu, M. N., Bellen, H., and Quiocho, F. A. (2000) Cell 100, 447-456]. Key features of the interaction surface between the FYVE and VHS domains of Hrs, involving helices 2 and 4 of the VHS domain, are conserved in the VHS domain of Tom1, even though Tom1 does not have a FYVE domain. We also compare the structures of the VHS domains of Tom1 and Hrs to the recently obtained structure of the ENTH domain of epsin-1 [Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P., and Brünger, A. T. (2000) J. Cell Biol. 149, 537-546]. Comparison of the two VHS domains and the ENTH domain reveals a conserved surface, composed of helices 2 and 4, that is utilized for protein-protein interactions. In addition, VHS domain-containing proteins are often localized to membranes. We suggest that the conserved positively charged surface of helix 3 in VHS and ENTH domains plays a role in membrane binding.     

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution,Obtained Using Small-Angle X-Ray Scattering

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed.     

Crystal structure of a 123 amino acids dimerization domain of Drosophila Caprin

Cytoplasmic activation/proliferation-associated protein (Caprin) proteins assume diverse functions in many important biological processes, including synaptic plasticity, stress response, innate immune response, and cellular proliferation. The Caprin family members are characterized by the presence of a highly conserved homologous region (HR1) at the N-terminus and arginine-glycine-rich (RGG) boxes at the C-terminus. We had previously determined the crystal structures of human Caprin-1 and Caprin-2 fragments corresponding to the C-terminal 2/3 of HR1. Both fragments adopt homodimeric structures. Based on sequence conservation, we speculated that all Caprin proteins should have similar homodimeric structures. Here we report the crystal structure of a fragment (residues 187-309) of Drosophila melanogaster Caprin (dCaprin). The dCaprin fragment adopts an all α-helical fold which self-associates to form a homodimer. The overall dCaprin homodimeric structure is similar to the Caprin-1 and Caprin-2 homodimeric structures. Most of the amino acids residues mediating homodimerization in the three structures are conserved among all Caprin family members. These structural and sequence data suggest that homodimerization through a conserved dimerization domain is a common structural feature of the Caprin protein family. The dimeric structures may also be involved in interaction with Caprin partners. Dimer formation creates a V-shape concave surface that may serve as a protein binding groove. The concave surfaces in Caprin-1, Caprin-2, and dCaprin should have different and specific binding partners due to the large difference in electrostatic potentials. We propose the existence of a multi-functional domain in Caprin proteins, which not only mediate homodimerization but also involve in interaction with specific Caprin partners.     

The Pilin Protein FimP from Actinomyces oris: Crystal Structure and Sequence Analyses

The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.     

Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive

Biogenesis of mitochondrial iron-sulfur (Fe/S) cluster proteins requires the interaction of multiple proteins with the highly conserved 14-kDa scaffold protein Isu, on which clusters are built prior to their transfer to recipient proteins. For example, the assembly process requires the cysteine desulfurase Nfs1, which serves as the sulfur donor for cluster assembly. The transfer process requires Jac1, a J-protein Hsp70 cochaperone. We recently identified three residues on the surface of Jac1 that form a hydrophobic patch critical for interaction with Isu. The results of molecular modeling of the Isu1-Jac1 interaction, which was guided by these experimental data and structural/biophysical information available for bacterial homologs, predicted the importance of three hydrophobic residues forming a patch on the surface of Isu1 for interaction with Jac1. Using Isu variants having alterations in residues that form the hydrophobic patch on the surface of Isu, this prediction was experimentally validated by in vitro binding assays. In addition, Nfs1 was found to require the same hydrophobic residues of Isu for binding, as does Jac1, suggesting that Jac1 and Nfs1 binding is mutually exclusive. In support of this conclusion, Jac1 and Nfs1 compete for binding to Isu. Evolutionary analysis revealed that residues involved in these interactions are conserved and that they are critical residues for the biogenesis of Fe/S cluster protein in vivo. We propose that competition between Jac1 and Nfs1 for Isu binding plays an important role in transitioning the Fe/S cluster biogenesis machinery from the cluster assembly step to the Hsp70-mediated transfer of the Fe/S cluster to recipient proteins.     

Substitutions in the redox-sensing PAS domain of the NifL regulatory protein define an inter-subunit pathway for redox signal transmission

The Per-ARNT-Sim (PAS) domain is a conserved α/β fold present within a plethora of signalling proteins from all kingdoms of life. PAS domains are often dimeric and act as versatile sensory and interaction modules to propagate environmental signals to effector domains. The NifL regulatory protein from Azotobacter vinelandii senses the oxygen status of the cell via an FAD cofactor accommodated within the first of two amino-terminal tandem PAS domains, termed PAS1 and PAS2. The redox signal perceived at PAS1 is relayed to PAS2 resulting in conformational reorganization of NifL and consequent inhibition of NifA activity. We have identified mutations in the cofactor-binding cavity of PAS1 that prevent 'release' of the inhibitory signal upon oxidation of FAD. Substitutions of conserved β-sheet residues on the distal surface of the FAD-binding cavity trap PAS1 in the inhibitory signalling state, irrespective of the redox state of the FAD group. In contrast, substitutions within the flanking A'α-helix that comprises part of the dimerization interface of PAS1 prevent transmission of the inhibitory signal. Taken together, these results suggest an inter-subunit pathway for redox signal transmission from PAS1 that propagates from core to the surface in a conformation-dependent manner requiring a flexible dimer interface.     

Crystal structure of human Gadd45 reveals an active dimer

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45, revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45 identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.     

Crucial motifs and residues in the extracellular loops influence the formation and specificity of connexin docking

Most of the early studies on gap junction (GJ) channel function and docking compatibility were on rodent connexins, while recent research on GJ channels gradually shifted from rodent to human connexins largely due to the fact that mutations in many human connexin genes are found to associate with inherited human diseases. The studies on human connexins have revealed some key differences from those found in rodents, calling for a comprehensive characterization of human GJ channels. Functional studies revealed that docking and formation of functional GJ channels between two hemichannels are possible only between docking-compatible connexins. Two groups of docking-compatible rodent connexins have been identified. Compatibility is believed to be due to their amino acid residue differences at the extracellular loop domains (E1 and E2). Sequence alignment of the E1 and E2 domains of all connexins known to make GJs revealed that they are highly conserved and show high sequence identity with human Cx26, which is the only connexin with near atomic resolution GJ structure. We hypothesize that different connexins have a similar structure as that of Cx26 at the E1 and E2 domains and use the corresponding residues in their E1 and E2 domains for docking. Based on the Cx26 GJ structure and sequence analysis of well-studied connexins, we propose that the E1-E1 docking interactions are staggered with each E1 interacting with two E1s on the docked connexon. The putative E1 docking residues are conserved in both docking-compatible and -incompatible connexins, indicating that E1 does not likely serve a role in docking compatibility. However, in the case of E2-E2 docking interactions, the putative docking residues are only conserved within the docking-compatible connexins, suggesting the E2 is likely to serve the function of docking compatibility. Docking compatibility studies on human connexins have attracted a lot of attention due to the fact that putative docking residues are mutational hotspots for several connexin-linked human diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.     

The N-terminal region of the bacterial DNA polymerase PolC features a pair of domains, both distantly related to domain V of the DNA polymerase III τ subunit

PolC is one of two essential replicative DNA polymerases in Bacillus subtilis and other Gram-positive bacteria. The 3D structure of PolC has recently been solved, yet it lacks the N-terminal region. For this PolC region of ～ 230 residues, both the structure and function are unknown. In the present study, using sensitive homology detection and comparative protein structure modeling, we identified, in this enigmatic region, two consecutive globular domains, PolC-NI and PolC-NII, which are followed by an apparently unstructured linker. Unexpectedly, we found that both domains are related to domain V of the τ subunit, which is part of the bacterial DNA polymerase III holoenzyme. Despite their common homology to τ, PolC-NI and PolC-NII exhibit very little sequence similarity to each other. This observation argues against simple tandem duplication within PolC as the origin of the two-domain structure. Using the derived structural models, we analyzed residue conservation and the surface properties of both PolC N-terminal domains. We detected a surface patch of positive electrostatic potential in PolC-NI and a hydrophobic surface patch in PolC-NII, suggesting their possible involvement in nucleic acid and protein binding, respectively. PolC is known to interact with the τ subunit, however, the region responsible for this interaction is unknown. We propose that the PolC N-terminus is involved in mediating the PolC-τ interaction and possibly also in binding DNA.     

Crystal structure of the UPF2-interacting domain of nonsense-mediated mRNA decay factor UPF1

UPF1 is an essential eukaryotic RNA helicase that plays a key role in various mRNA degradation pathways, notably nonsense-mediated mRNA decay (NMD). In combination with UPF2 and UPF3, it forms part of the surveillance complex that detects mRNAs containing premature stop codons and triggers their degradation in all organisms studied from yeast to human. We describe the 3 A resolution crystal structure of the highly conserved cysteine-histidine-rich domain of human UPF1 and show that it is a unique combination of three zinc-binding motifs arranged into two tandem modules related to the RING-box and U-box domains of ubiquitin ligases. This UPF1 domain interacts with UPF2, and we identified by mutational analysis residues in two distinct conserved surface regions of UPF1 that mediate this interaction. UPF1 residues we identify as important for the interaction with UPF2 are not conserved in UPF1 homologs from certain unicellular parasites that also appear to lack UPF2 in their genomes.