Recognition of ubiquitin and polyubiquitin chains by ubiquitin-binding domains (UBDs) is vital for ubiquitin-mediated signaling pathways. The endoplasmic reticulum resident RING finger ubiquitin ligase (E3) gp78 regulates critical proteins via the ubiquitin-proteasome system to maintain cellular homeostasis and includes a UBD known as the CUE domain, which is essential for function. A probable role of this domain is to recognize ubiquitin modified substrates, enabling gp78 to assemble polyubiquitin chains on these substrates and mark them for degradation. Here, we report the molecular details of the interaction of gp78CUE domain with ubiquitin and diubiquitin. The gp78CUE domain exhibits a well-defined set of interactions with ubiquitin and a dynamic, promiscuous interaction with diubiquitin chains. This leads to a model where the CUE domain functions to both facilitate substrate binding and enables switching between adjacent ubiquitin molecules of a growing chain to facilitate processivity in ubiquitination.
RCK domains control activity of a variety of K+ channels and transporters through binding of cytoplasmic ligands. To gain insight toward mechanisms of RCK domain activation, we solved the structure of the RCK domain from the Ca2+-gated K+ channel, MthK, bound with Ba2+, at 3.1 Å resolution. The Ba2+-bound RCK domain was assembled as an octameric gating ring, as observed in structures of the full-length MthK channel, and shows Ba2+ bound at several positions. One of the Ba2+ sites, termed C1, overlaps with a known Ca2+-activation site, determined by residues D184 and E210. Functionally, Ba2+ can activate reconstituted MthK channels as observed in electrophysiological recordings, whereas Mg2+ (up to 100 mM) was ineffective. Ba2+ activation was abolished by the mutation D184N, suggesting that Ba2+ activates primarily through the C1 site. Our results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating.
K channel; barium; lipid bilayer; allosteric
HCN channels and their modulation by cAMP play a key role in cardiac pacemaking. In this issue of Structure, Xu and colleagues reveal that an arrhythmia-causing mutation of an HCN channel weakens cAMP binding to the channel by altering the local structure of its entry-exit pathway.
Single particle electron microscopy (EM) is a powerful tool for studying the structures of large biological molecules. However, the achievable resolution does not always allow for direct recognition of individual protein domains. Labels that can be visualized by EM have been developed for protein termini, but tagging internal domains remains a challenge. We describe a robust strategy for determining the position of internal sites within EM maps, termed Domain Localization by RCT Sampling (DOLORS). DOLORS uses monovalent streptavidin added post-translationally to tagged sites in the target protein. Internal labels generally display less conformational flexibility than terminal labels, providing more precise positional information. Automated methods are used to rapidly generate assemblies of unique 3D models allowing the attachment sites of labeled domains to be accurately identified and thus provide an overall architectural map of the molecule.
With multi-drug resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug-targets are becoming an urgent need. Traditional β-lactam antibiotics that disrupt the D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on β-lactam insensitive L,D-transpeptidases for biosynthesis and maintenance of their peptidoglycan layer. This reliance plays a major role in drug-resistance and persistence of Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the Mtb L,D-transpeptidase LdtMt2 containing a bound peptidoglycan fragment, reported here, provides information about catalytic site organization as well as substrate recognition by the enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic mechanism for LdtMt2 in which both acyl-acceptor and acyl-donor substrates reach the catalytic site from the same, rather than different, entrances. Together, this information provides vital insights for the development of novel drugs targeting this validated yet unexploited enzyme.
c-Jun N-terminal (JNK) family kinases have a common peptide-docking site used by upstream activating kinases, substrates, scaffold proteins, and phosphatases, where the ensemble of bound proteins determines signaling output. Although there are many JNK structures, little is known about mechanisms of allosteric regulation between the catalytic and peptide-binding sites, and the activation loop, whose phosphorylation is required for catalytic activity. Here, we compare three structures of unliganded JNK3 bound to different peptides. These were compared as a class to structures that differ in binding of peptide, small molecule ligand, or conformation of the kinase activation loop. Peptide binding induced an inhibitory interlobe conformer that was reversed by alterations in the activation loop. Structure class analysis revealed the subtle structural mechanisms for allosteric signaling between the peptide-binding site and activation loop. Biochemical data from isothermal calorimetry, fluorescence energy transfer, and enzyme inhibition demonstrated affinity differences among the three peptides that were consistent with structural observations.
Cryo-electron microscopy (cryo-EM) is a powerful technique for 3D structure determination of protein complexes by averaging information from individual molecular images. The resolutions that can be achieved with single-particle cryo-EM are frequently limited by inaccuracies in assigning molecular orientations based solely on 2D projection images. Tomographic data collection schemes, however, provide powerful constraints that can be used to more accurately determine molecular orientations necessary for 3D reconstruction. Here, we propose “constrained single-particle tomography” as a general strategy for 3D structure determination in cryo-EM. A key component of our approach is the effective use of images recorded in tilt series to extract high-resolution information and correct for the contrast transfer function. By incorporating geometric constraints into the refinement to improve orientational accuracy of images, we reduce model bias and overrefinement artifacts and demonstrate that protein structures can be determined at resolutions of ~8 Å starting from low-dose tomographic tilt series.
RIG-I is a cytosolic sensor of viral RNA, comprised of two N-terminal CARDs followed by helicase and C-terminal regulatory domains (helicase-CTD). Viral RNA binds to the helicase-CTD and “exposes” the CARDs for downstream signaling. The role of the second CARD (CARD2) is essential as RIG-I activation requires dephosphorylation of Thr170 followed by ubiquitination at Lys172. Here, we present the solution structure and dynamics of human RIG-I CARD2. Surprisingly, we find that Thr170 is mostly buried. Parallel studies on the phosphomimetic T170E mutant suggest that the loss of function upon Thr170 phosphorylation is likely associated with changes in the CARD1–CARD2 interface that may prevent Lys172 ubiquitination and/or binding to free K63-linked polyubiquitin. We also demonstrate a strong interaction between CARD2 and the helicase-CTD, and show that mutations at the interface result in constitutive activation of RIG-I. Collectively, our data suggests a close interplay between phosphorylation, ubiquitination, and activation of human RIG-I, all mediated by CARD2.
Neisseria gonorrhoeae is an obligate human pathogen that can escape immune surveillance through antigenic variation of surface structures such as pili. A G-quadruplex-forming (G4) sequence (5´-G3TG3TTG3TG3) located upstream of the N. gonorrhoeae pilin expression locus (pilE) is necessary for initiation of pilin antigenic variation, a recombination-based, high-frequency, diversity-generation system. We have determined NMR-based structures of the all-parallel-stranded monomeric and novel 5´-end-stacked dimeric pilE G-quadruplexes in monovalent cation-containing solutions. We demonstrate that the three-layered all-parallel-stranded monomeric pilE G-quadruplex containing single residue double-chain-reversal loops, that can be modeled without steric clashes into the three-nucleotide DNA-binding site of RecA, binds and promotes E. coli RecA mediated strand exchange in vitro. We discuss how interactions between RecA and monomeric pilE G-quadruplex could facilitate the specialized recombination reactions leading to pilin diversification.
Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans.
A distinctive mechanism for ubiquitin (Ub) ligation has recently been proposed for the RING1-IBR-RING2 (RBR) family of E3s: an N-terminal RING1 domain recruits a thioester-linked intermediate complex between Ub and the E2 UbcH7, and a structurally unique C-terminal RING2 domain displays a catalytic cysteine required for Ub ligation. To obtain insights into RBR E3s, we determined the crystal structure of the Human Homolog of Ariadne (HHARI), which reveals the individual RING1, IBR, and RING2 domains embedded in superdomains involving sequences specific to the Ariadne RBR subfamily. The central IBR is flanked on one side by RING1, which is exposed and binds UbcH7. On the other side, a C-terminal autoinhibitory “Ariadne domain” masks the RING2 active site. Insights into RBR E3 mechanisms are provided by structure-based mutations that indicate distinct steps of relief from autoinhibition, Ub transfer from E2 to HHARI, and ligation from the HHARI cysteine to a terminal acceptor.
Pygo proteins promote Armadillo- and β-catenin-dependent transcription, by relieving Groucho-dependent repression of Wnt targets. Their PHD fingers bind histone H3 tail methylated at lysine 4, and to the HD1 domain of their Legless/BCL9 cofactors, linking Pygo to Armadillo/β-catenin. Intriguingly, fly Pygo orthologs exhibit a tryptophan > phenylalanine substitution in their histone pocket-divider which reduces their affinity for histones. Here, we use X-ray crystallography and NMR, to discover a conspicuous groove bordering this phenylalanine in the Drosophila PHD-HD1 complex—a semi-aromatic cage recognizing asymmetrically methylated arginine 2 (R2me2a), a chromatin mark of silenced genes. Our structural model of the ternary complex reveals a distinct mode of dimethylarginine recognition, involving a polar interaction between R2me2a and its groove, the structural integrity of which is crucial for normal tissue patterning. Notably, humanized fly Pygo derepresses Notch targets, implying an inherent Notch-related function of classical Pygo orthologs, disabled in fly Pygo, which thus appears dedicated to Wnt signaling.
•Coadapted mutations in fly Pygo PHD fingers alter their histone-binding surface•A semi-aromatic groove in fly Pygo embeds dimethylated arginine 2 of histone H3•Structural integrity of the arginine 2 groove is required for tissue patterning•Humanized Drosophila Pygo derepresses Notch targets
Miller et al. show that coadapted mutations in the Drosophila Pygo PHD finger alter its histone-binding surface to contain a novel semi-aromatic groove that embeds asymmetrically methylated arginine 2 of the histone H3 tail, a chromatin mark of silenced genes.
RLIP76 is an effector for Ral small GTPases, which in turn lie downstream of the master regulator Ras. Evidence is growing that Ral and RLIP76 play a role in tumorigenesis, invasion, and metastasis. RLIP76 contains both a RhoGAP domain and a Ral binding domain (GBD) and is, therefore, a node between Ras and Rho family signaling. The structure of the RhoGAP-GBD dyad reveals that the RLIP76 RhoGAP domain adopts a canonical RhoGAP domain structure and that the linker between the two RLIP76 domains is structured, fixing the orientation of the two domains and allowing RLIP76 to interact with Rho-family GTPases and Ral simultaneously. However, the juxtaposed domains do not influence each other functionally, suggesting that the RLIP76-Ral interaction controls cellular localization and that the fixed orientation of the two domains orientates the RhoGAP domain with respect to the membrane, allowing it to be perfectly poised to engage its target G proteins.
•The structure of the RLIP76 RhoGAP-Ral binding domain dyad has been solved•The interdomain linker contacts both domains and fixes their orientation•The RhoGAP domain is a poor GAP for Cdc42 and Rac1 in vitro•Ral and Rho family proteins can interact simultaneously with RLIP76
RLIP76 is an effector for Ral small GTPases that contains a RhoGAP domain adjacent to a Ral-binding domain. Rajasekar et al. solve a structure and reveal that the linker between the two domains is structured, fixing their orientation. This orientation allows them to engage their target G proteins.
ARC1172 is a 41-mer DNA aptamer selected to bind the A1 domain of von Willebrand factor (VWF). A derivative of ARC1172 with modifications to increase intravascular survival inhibits carotid artery thrombosis in a Cynomolgus macaque model and inhibits VWF-dependent platelet aggregation in humans, suggesting that such aptamers may be useful to prevent or treat thrombosis. In the crystal structure of a VWF A1-ARC1172 complex, the aptamer adopts a three-stem structure of mainly B-form DNA with three noncanonical base pairs and 9 unpaired residues, 6 of which are stabilized by base-base or base-deoxyribose stacking interactions. The aptamer-protein interface is characterized by cation-π interactions involving Arg, Lys and Gln residues, often stabilized by H-bonds with adjacent bases. The ARC1172 binding site on the A1 domain overlaps with that of botrocetin and clashes with glycoprotein Ibα binding at an adjacent site, which accounts for the antithrombotic activity of ARC1172 and related aptamers.
In K+ channels, rearrangements of the pore outer-vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggest these movements to be modest in magnitude. Here, we show that under physiological conditions, the KcsA outer-vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd2+ enhances the rate of C-type inactivation in an outer-vestibule cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K+ channels demonstrate that these Cd2+ metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd2+-bound Y82C-KcsA in the closed state, together with EPR distance measurements in the KcsA outer-vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation.
Ionotropic glutamate receptors (iGluRs) harbor two domains in their extracellular region: the membrane-proximal ligand-binding domain (LBD) and the distal N-terminal domain (NTD). These are involved in signal sensing: the LBD binds L-glutamate, which activates the receptor channel. Ligand-binding to NTD modulates channel function in the NMDA receptor subfamily of iGluRs, which has not been observed for the AMPA receptor (AMPAR) subfamily to date. Structural data suggest that AMPAR NTDs are packed into tight dimers and have lost their signaling potential. Here we assess NTD dynamics from both subfamilies using a variety of computational tools. We describe the conformational motions which underly NMDAR NTD allosteric signaling. Unexpectedly, AMPAR NTDs are capable of undergoing similar dynamics; although dimerization imposes restrictions, the two subfamilies sample similar, interconvertible conformational subspaces. Finally, we solve the crystal structure of AMPAR GluA4 NTD, and combined with all-atom molecular dynamics simulations we characterize regions pivotal for an as yet unexplored dynamic spectrum of AMPAR NTDs.
Glutamate receptor N-terminal Domain; GluA4 NTD structure; Intrinsic dynamics; Clamshell-like motions; NTD allostery
MTERF4 is the first MTERF family member shown to bind RNA, and plays an essential role as a regulator of ribosomal biogenesis in mammalian mitochondria. It forms a complex with the rRNA methyltransferase NSUN4 and recruits it to the large ribosomal subunit. In this paper, we characterize the interaction between both proteins, demonstrate that MTERF4 strongly stimulates the specificity of NSUN4 during in vitro methylation experiments and present the 2.0 Å resolution crystal structure of the MTERF4:NSUN4 protein complex, lacking 48 residues of the MTERF4 C-terminal acidic tail, bound to S-adenosyl-L-methionine, revealing the nature of the interaction between both proteins and the structural conservation of the most divergent of the human MTERF family members. Moreover, the structure suggests a model for RNA binding by the MTERF4:NSUN4 complex, providing insight into the mechanism by which an MTERF family member facilitates rRNA methylation.
Centrioles are key microtubule polarity determinants. Centriole duplication is tightly controlled to prevent cells from developing multipolar spindles, a situation that promotes chromosomal instability. A conserved component in the duplication pathway is Plk4, a polo kinase family member that localizes to centrioles in M/G1. To limit centriole duplication, Plk4 levels are controlled through trans-autophosphorylation that primes ubiquitination. In contrast to Plks 1–3, Plk4 possesses a unique central region called the “cryptic polo box”. Here, we present the crystal structure of this region at 2.3 Å resolution. Surprisingly, the structure reveals two tandem, homodimerized polo boxes, PB1-PB2, that form a unique, winged architecture. The full PB1-PB2 cassette is required for binding the centriolar protein Asterless as well as robust centriole targeting. Thus, with its C-terminal polo box (PB3), Plk4 has a triple polo box architecture that facilitates oligomerization, targeting, and promotes trans-autophosphorylation, limiting centriole duplication to once per cell cycle.
Vacuolar ATPases (V-ATPases) are multisubunit rotary motor proton pumps that function to acidify subcellular organelles in all eukaryotic organisms. V-ATPase is regulated by a unique mechanism that involves reversible dissociation into V1-ATPase and Vo proton channel, a process that involves breaking of protein interactions mediated by subunit C, the cytoplasmic domain of subunit 'a' and three 'peripheral stalks', each made of a heterodimer of E and G subunits. Here we present crystal structures of a yeast V-ATPase heterotrimeric complex composed of EG heterodimer and the head domain of subunit C (Chead). The structures show EG heterodimer folded in a non-canonical coiled coil that is stabilized at its N-terminal ends by binding to Chead. The coiled coil is disrupted by a bulge of partially unfolded secondary structure in subunit G and we speculate that this unique feature in the eukaryotic V-ATPase peripheral stalk may play an important role in enzyme structure and regulation by reversible dissociation.
DNA polymerase and substrate conformational changes are essential for high fidelity DNA synthesis. Structures of DNA polymerase (pol) β in complex with DNA show the enzyme in an ‘open’ conformation. Subsequent to binding the nucleotide, the polymerase ‘closes’ around the nascent base pair with two metals positioned for chemistry. However, structures of substrate/active site intermediates prior to closure are lacking. By destabilizing the closed complex, we determined unique ternary complex structures of pol β with correct and incorrect incoming nucleotides bound to the open conformation. These structures reveal Watson-Crick hydrogen bonding is assessed upon initial complex formation. Importantly, novel nucleotidebound states representing intermediate metal coordination states occur with active site assembly. The correct, but not incorrect, nucleotide maintains Watson-Crick hydrogen bonds during interconversion of these states. These structures indicate that the triphosphate of the incoming nucleotide undergoes rearrangement prior to closure providing an opportunity to deter misinsertion and increase fidelity.
Lactose permease of Escherichia coli (LacY) catalyzes symport of a galactopyranoside and an H+ via an alternating access mechanism. The transition from an inward- to an outward-facing conformation of LacY involves sugar-release followed by deprotonation. Because the transition depends intimately upon the dynamics of LacY in a bilayer environment, molecular dynamics (MD) simulations may be the only means of following the accompanying structural changes in atomic detail. We describe here MD simulations of wild-type apo LacY in phosphatidylethanolamine (POPE) lipids that features two protonation states of the critical Glu325. While the protonated system displays configurational stability, deprotonation of Glu325 causes significant structural rearrangements that bring into proximity sidechains important for H+ translocation and sugar binding and closes the internal cavity. Moreover, protonated LacY in phosphatidylcholine (DMPC) lipids shows that the observed dynamics are lipid-dependent. Together, the simulations describe early dynamics of the inward-to-outward transition of LacY that agree well with experimental data.
lactose permease; protonation states; protein-lipid interactions; MD simulations
Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B (RANK) are members of the TNFR superfamily that regulate osteoclast formation and function by competing for RANK ligand (RANKL). RANKL promotes osteoclast development through RANK activation, while OPG inhibits this process by sequestering RANKL. For comparison, we solved crystal structures of RANKL with RANK, and RANKL with OPG. Complementary biochemical and functional studies reveal that the monomeric cytokine-binding region of OPG binds RANKL with ~500 fold higher affinity than RANK, and inhibits RANKL-stimulated osteoclastogenesis ~150 times more effectively, in part because the binding cleft of RANKL makes unique contacts with OPG. Several side chains as well as the C-D and D-E loops of RANKL occupy different orientations when bound to OPG versus RANK. High affinity OPG binding requires a 90s-loop Phe residue that is mutated in juvenile Paget’s disease. These results suggest cytokine plasticity may help to fine tune specific TNF-family cytokine/receptor pair selectivity.
Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry. CPAP, a centriolar protein essential for microtubule recruitment, features a C-terminal domain of unknown structure, the G-box. A missense mutation in the G-box reduces affinity for the centriolar shuttling protein STIL and causes primary microcephaly. Here, we characterize the molecular architecture of CPAP and determine the G-box structure alone and in complex with a STIL fragment. The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies. Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex. We propose that CPAP acts as a horizontal “strut” that joins the centriolar scaffold with microtubules, whereas G-box domains form perpendicular connections.
•CPAP features a long dimeric parallel coiled coil and a C-terminal domain (G-box)•The G-box adopts an elongated structure with a single β sheet•G-box domains form long fibrils with periodicity equivalent to centriolar spacing•STIL features a conserved proline-rich motif that is important for CPAP binding
Hatzopoulos et al. describe the molecular architecture of the essential centriolar protein CPAP and the structure of its G-box domain. G-box comprises a single β sheet and can form soluble amyloid-like fibrils. The model of amyloid-like fibrils suggests a structural role in centriole assembly for CPAP.
We report the solution NMR structure of a designed dimetal-binding protein, di-Zn(II) DFsc, along with a secondary refinement step employing molecular dynamics techniques. Calculation of the initial NMR structural ensemble by standard methods led to distortions in the metal-ligand geometries at the active site. Unrestrained molecular dynamics using a non-bonded force field for the metal shell, followed by quantum mechanical/molecular mechanical dynamics of DFsc, were used to relax local frustrations at the dimetal site that were apparent in the initial NMR structure and provide a more realistic description of the structure. The MD model is consistent with NMR restraints, and in good agreement with the structural and functional properties expected for DF proteins. This work demonstrates that NMR structures of metalloproteins can be further refined using classical and first-principles molecular dynamics methods in the presence of explicit solvent to provide otherwise unavailable insight into the geometry of the metal center.
Intrinsically disordered protein (IDP)-mediated interactions are often characterized by low affinity but high specificity. These traits are essential in signaling and regulation that require reversibility. Enterohaemorrhagic Escherichia coli (EHEC) exploit this situation by commandeering host cytoskeletal signaling to stimulate actin assembly beneath bound bacteria, generating ‘pedestals’ that promote intestinal colonization. EHEC translocates into the host cell two proteins, EspFU and Tir, which form a complex with the host protein IRTKS. The interaction of this complex with N-WASP triggers localized actin polymerization. We show that EspFU is an IDP that contains a transiently α-helical N-terminus and dynamic C-terminus. Our structure shows that single EspFU repeat is capable of forming a high-affinity trimolecular complex with N-WASP and IRTKS. We demonstrate that bacterial and cellular ligands interact with IRTKS SH3 in a similar fashion but the bacterial protein has evolved to outcompete cellular targets by utilizing a tryptophan switch that offers superior binding affinity enabling EHEC-induced pedestal formation.