Structural insights into the interaction of the crenarchaeal chromatin protein Cren7 with DNA

Cren7, a newly found chromatin protein, is highly conserved in the Crenarchaeota. The protein shows higher affinity for double‐stranded DNA than for single‐stranded DNA, constrains negative DNA supercoils in vitro and is associated with genomic DNA in vivo. Here we report the crystal structures of the Cren7 protein from Sulfolobus solfataricus in complex with two DNA sequences. Cren7 binds in the minor groove of DNA and causes a single‐step sharp kink in DNA (～53°) through the intercalation of the hydrophobic side chain of Leu28. Loop β3‐β4 of Cren7 undergoes a significant conformational change upon binding of the protein to DNA, suggesting its critical role in the stabilization of the protein–DNA complex. The roles of DNA‐contacting amino acid residues in stabilizing the Cren7–DNA interaction were examined by mutational analysis. Structural comparison of Cren7‐DNA complexes with Sac7d‐DNA complexes reveals significant differences between the two proteins in DNA binding surface, suggesting that Cren7 and Sul7d serve distinct functions in chromosomal organization.     

Structural insights into the interaction of insulin-like growth factor 2 with IGF2R domain 11

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.     

Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding.     

Structural insights into the EB1-APC interaction

EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. We find that EB1 is a stable dimer with a parallel coiled coil and show that dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 microM affinity. The conserved APC Ile2805-Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789-Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC-EB1 is post-translationally regulated in cells. Our findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms.     

Structural insights into RNA recognition by RIG-I

Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved "helicase" domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomechanical function of this protein family and other ATPases more broadly.     

Structural insights into the U-box,a domain associated with multi-ubiquitination

The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val-->Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.     

New insights into the interaction of ribosomal protein L1 with RNA

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.     

Structural insights into the interaction of ROCKI with the switch regions of RhoA

The Rho-ROCK pathway modulates the phosphorylation level of a variety of important signaling proteins and is thereby involved in miscellaneous cellular processes including cell migration, neurite outgrowth, and smooth muscle contraction. The observation of the involvement of the Rho-ROCK pathway in tumor invasion and in diseases such as hypertension and bronchial asthma makes it an interesting target for drug development. We herein present the crystal structure of the complex between active RhoA and the Rho-binding domain of ROCKI. The Rho-binding domain structure forms a parallel alpha-helical coiled-coil dimer and, in contrast to the published Rho-protein kinase N structure, binds exclusively to the switch I and II regions of the guanosine 5'-(beta,gamma-imido)triphosphate-bound RhoA. The switch regions of two different RhoA molecules form a predominantly hydrophobic patch, which is complementarily bound by two identical short helices of 13 residues (amino acids 998-1010). The identified ROCK-binding site of RhoA strikingly supports the assumption of a common consensus-binding site for effector recognition.     

Structural insights into the cytoplasmic domain of a human BK channel

The large conductance, voltage- and Ca(2+) -activated K(+) (BK or Slo1) channel is widely expressed in mammalian cells/tissues (i.e. neurons, skeletal and smooth muscles, exocrine cells, the inner ear) and regulates action potential firing, muscle contraction and secretion. The large ionic conductance and unusual, dual stimulus-driven gating behavior of this channel have long intrigued membrane biophysicists, and recent structure/function analyses have provided increasingly detailed insights into the molecular bells and whistles that regulate BK channel activity. Now, in two complementary articles published by the groups of Rod MacKinnon and Youxing Jiang, high resolution x-ray crystal structures of the human BK channel's large cytoplasmic domain have been solved in both the absence and presence of bound Ca(2+), conditions which would presumably promote the resting and activated conformations of this large domain. Given the regulatory importance of the cytosolic domain on BK channel gating, these experimentally determined structures reveal a number of key insights, including: 1) the physical arrangement and interactions of the tandem RCK1 and RCK2 domains within a single channel subunit, 2) the assembly of the four large cytoplasmic domains into a symmetric, tetrameric complex, 3) the formation of the channel's gating ring structure, based on the assembly of the individual RCK1 and 2 domains, and 4) the structural elements underlying the regions critical for divalent metal ion binding (i.e. Ca (2+) and Mg (2+)) and their potential influence on conduction pore.     

Structural insights into the GTPase domain of Escherichia coli MnmE protein

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well as on the formation of a MnmE transition state mimic. GTP hydrolysis by MnmE, but not GTP binding or formation of a complex with mant-GDP and aluminium fluoride, is impaired at acidic pH, suggesting that the chemistry of the transition state mimic is different to that of the true transition state, and that some residue(s), critical for GTP hydrolysis, is severely affected by low pH. We use a nuclear magnetic resonance (NMR)-based approach to get insights into the MnmE structure and properties. The combined use of NMR restraints and homology structural information allowed the determination of the MnmE G-domain structure in its free form. Chemical shift structure-based prediction provided a good basis for structure refinement and validation. Our data support that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis, although Arg252 may play a role in stabilization of the transition state.     

Structural and functional insights into the B30.2/SPRY domain

The B30.2/SPRY domain is present in approximately 700 eukaryotic (approximately 150 human) proteins, including medically important proteins such as TRIM5alpha and Pyrin. Nonetheless, the functional role of this modular domain remained unclear. Here, we report the crystal structure of an SPRY-SOCS box family protein GUSTAVUS in complex with Elongins B and C, revealing a highly distorted two-layered beta-sandwich core structure of its B30.2/SPRY domain. Ensuing studies identified one end of the beta-sandwich as the surface interacting with an RNA helicase VASA with a 40 nM dissociation constant. The sequence variation in TRIM5alpha responsible for HIV-1 restriction and most of the mutations in Pyrin causing familial Mediterranean fever map on this surface, implicating the corresponding region in many B30.2/SPRY domains as the ligand-binding site. The amino acids lining the binding surface are highly variable among the B30.2/SPRY domains, suggesting that these domains are protein-interacting modules, which recognize a specific individual partner protein rather than a consensus sequence motif.     

Structure of MST2 SARAH domain provides insights into its interaction with RAPL

The STE20 kinases MST1 and MST2 are key players in mammalian Hippo pathway. The SARAH domains of MST1/2 act as a platform to mediate homodimerization and hetero-interaction with a range of adaptors including RASSFs and Salvador, which also possess SARAH domains. Here, we determined the crystal structure of human MST2 SARAH domain, which forms an antiparallel homodimeric coiled coil. Structural comparison indicates that SARAH domains of different proteins may utilize a shared dimerization module to form homodimer or heterodimer. Structure-guided mutational study identified specific interface residues critical for MST2 homodimerization. MST2 mutations disrupting its homodimerization also impaired its hetero-interaction with RAPL (also named RASSF5 and NORE1), which is mediated by their SARAH domains. Further biochemical and cellular assays indicated that SARAH domain-mediated homodimerization and hetero-interaction with RAPL are required for full activation of MST2 and therefore apoptotic functions in T cells.     

Structural insights into RNA recognition and activation of RIG-I-like receptors

RIG-I like receptors (RLR) that recognize non-self RNA play critical roles in activating host innate immune pathways in response to viral infections. Not surprisingly, RLRs and their associated signaling networks are also targeted by numerous antagonists that facilitate viral pathogenesis. Although the role of RLRs in orchestrating antiviral signaling has been recognized for some time, our knowledge of the complex regulatory mechanisms that control signaling through these key molecules is incomplete. A series of recent structural studies shed new light into the structural basis for dsRNA recognition and activation of RLRs. Collectively, these studies suggest that the repression of RLRs is facilitated by a cis element that makes multiple contacts with domains within the helicase and that RNA binding initiated by the C-terminal RNA binding domain is important for ATP hydrolysis and release of the CARD domain containing signaling module from the repressed conformation. These studies also highlight potential differences between RIG-I and MDA5, two RLR members. Together with previous studies, these new results bring us a step closer to uncovering the complex regulatory process of a key protein that protects host cells from invading pathogens.