Solution structure of the VEGF-binding domain of Flt-1: comparison of its free and bound states.

The extracellular portion of the VEGF and PlGF receptor, Flt-1 (or VEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1D2) is necessary and sufficient for high affinity VEGF binding. The 1.7 A resolution crystal structure of Flt-1D2 bound to VEGF revealed that this domain is a member of the I-set of the immunoglobulin superfamily, but has several unusual features including a region near the N terminus that bulges away from the domain rather than pairing with the neighboring beta-strand. Some of the residues in this region make contact with VEGF, raising the possibility that this bulge could be a consequence of VEGF binding and might not be present in the absence of ligand. Here we report the three-dimensional structure of Flt-1D2 in its uncomplexed form determined by NMR spectroscopy. A semi-automated method for NOE assignment that takes advantage of the previously solved crystal structure was used to facilitate rapid analysis of the 3D NOESY spectra. The solution structure is very similar to the previously reported VEGF-bound crystal structure; the N-terminal bulge is present, albeit in a different conformation. We also report the 2.7 A crystal structure of Flt-1D2 in complex with VEGF solved in a different crystal form that reveals yet another conformation for the N-terminal bulge region. (1)H-(15)N heteronuclear NOEs indicate this region is flexible in solution; the crystal structures show that this region is able to adopt more than one conformation even when bound to VEGF. Thus, VEGF-binding is not accompanied by significant structural change in Flt-1D2, and the unusual structural features of Flt-1D2 are an intrinsic property of this domain.     

The structure of MSK1 reveals a novel autoinhibitory conformation for a dual kinase protein

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.     

The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment

Kinetochores are multicomponent assemblies that connect chromosomal centromeres to mitotic-spindle microtubules. The Ndc80 complex is an essential core element of kinetochores, conserved from yeast to humans. It is a rod-like assembly of four proteins- Ndc80p (HEC1 in humans), Nuf2p, Spc24p and Spc25p. We describe here the crystal structure of the most conserved region of HEC1, which lies at one end of the rod and near the N terminus of the polypeptide chain. It folds into a calponin-homology domain, resembling the microtubule-binding domain of the plus-end-associated protein EB1. We show that an Ndc80p-Nuf2p heterodimer binds microtubules in vitro. The less conserved, N-terminal segment of Ndc80p contributes to the interaction and may be a crucial regulatory element. We propose that the Ndc80 complex forms a direct link between kinetochore core components and spindle microtubules.     

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform ¹³C and ¹⁵N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.     

The Mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37)

Recruitment of protein kinase clients to the Hsp90 chaperone involves the cochaperone p50(cdc37) acting as a scaffold, binding protein kinases via its N-terminal domain and Hsp90 via its C-terminal region. p50(cdc37) also has a regulatory activity, arresting Hsp90's ATPase cycle during client-protein loading. We have localized the binding site for p50(cdc37) to the N-terminal nucleotide binding domain of Hsp90 and determined the crystal structure of the Hsp90-p50(cdc37) core complex. Dimeric p50(cdc37) binds to surfaces of the Hsp90 N-domain implicated in ATP-dependent N-terminal dimerization and association with the middle segment of the chaperone. This interaction fixes the lid segment in an open conformation, inserts an arginine side chain into the ATP binding pocket to disable catalysis, and prevents trans-activating interaction of the N domains.     

Structure, dynamics, and Hck interaction of full-length HIV-1 Nef

Nef is an HIV accessory protein that plays an important role in the progression of disease after viral infection. It interferes with numerous signaling pathways, one of which involves serine/threonine kinases. Here, we report the results of an NMR structural investigation on full-length Nef and its interaction with the entire regulatory domain of Hck (residues 72-256; Hck32L). A helical conformation was found at the N-terminus for residues 14-22, preceding the folded core domain. In contrast to the previously studied truncated Nef (Nef Δ1-39), the full-length Nef did not show any interactions of Trp57/Leu58 with the hydrophobic patch formed by helices α1 and α2. Upon Hck32L binding, the N-terminal anchor domain as well as the well-known SH3-binding site of Nef exhibited significant chemical shift changes. Upon Nef binding, resonance changes in the Hck spectrum were confined mostly to the SH3 domain, with additional effects seen for the connector between SH3 and SH2, the N-terminal region of SH2 and the linker region that contains the regulatory polyproline motif. The binding data suggest that in full-length Nef more than the core domain partakes in the interaction. The solution conformation of Hck32L was modeled using RDC data and compared with the crystal structure of the equivalent region in the inactivated, full-length Hck, revealing a notable difference in the relative orientations of the SH3 and SH2 domains. The RDC-based model combined with (15)N backbone dynamics data suggest that Hck32L adopts an open conformation without binding of the polyproline motif in the linker to the SH3 domain.     

Solution structure of the capsid protein from the human T-cell leukemia virus type-I.

The solution structure of the capsid protein (CA) from the human T-cell leukemia virus type one (HTLV-I), a retrovirus that causes T-cell leukemia and HTLV-I-associated myelopathy in humans, has been determined by NMR methods. The protein consists of independent N and C-terminal domains connected by a flexible linker. The domains are structurally similar to the N-terminal "core" and C-terminal "dimerization" domains, respectively, of the human immunodeficiency virus type one (HIV-1) and equine infectious anemia virus (EIAV) capsid proteins, although several important differences exist. In particular, hydrophobic residues near the major homology region are partially buried in HTLV-I CA, which is monomeric in solution, whereas analogous residues in HIV-1 and EIAV CA project from the C-terminal domain and promote dimerization. These differences in the structure and oligomerization state of the proteins appear to be related to, and possibly controlled by, the oxidation state of conserved cysteine residues, which are reduced in HTLV-I CA but form a disulfide bond in the HIV-1 and EIAV CA crystal structures. The results are consistent with an oxidative capsid assembly mechanism, in which CA oligomerization or maturation is triggered by disulfide bo nd formation as the budding virus enters the oxidizing environment of the bloodstream.     

The structure of G protein-coupled receptor kinase (GRK)-6 defines a second lineage of GRKs

We describe the 2.6-A crystal structure of human G protein-coupled receptor kinase (GRK)-6, a key regulator of dopaminergic signaling and lymphocyte chemotaxis. GRK6 is a member of the GRK4 subfamily of GRKs, which is represented in most, if not all, metazoans. Comparison of GRK6 with GRK2 confirms that the catalytic core of all GRKs consists of intimately associated kinase and regulator of G protein signaling (RGS) homology domains. Despite being in complex with an ATP analog, the kinase domain of GRK6 remains in an open, presumably inactive conformation, suggesting that G protein-coupled receptors activate GRKs by inducing kinase domain closure. The structure reveals a putative phospholipid-binding site near the N terminus of GRK6 and structural elements within the kinase substrate channel that likely influence G protein-coupled receptor access and specificity. The crystalline GRK6 RGS homology domain forms an extensive dimer interface using conserved hydrophobic residues distinct from those in GRK2 that bind Galpha(q), although dimerization does not appear to occur in solution and is not required for receptor phosphorylation.     

Crystal structure of the amino-terminal coiled-coil domain of the APC tumor suppressor

Coiled coils serve as dimerization domains for a wide variety of proteins, including the medically important oligomeric tumor suppressor protein, APC. Mutations in the APC gene are associated with an inherited susceptibility to colon cancer and with approximately 75 % of sporadic colorectal tumors. To define the basis for APC pairing and to explore the anatomy of dimeric coiled coils, we determined the 2.4 A resolution X-ray crystal structure of the N-terminal dimerization domain of APC. The peptide APC-55, encompassing the heptad repeats in APC residues 2-55, primarily forms an alpha-helical, coiled-coil dimer with newly observed core packing features. Correlated asymmetric packing of four core residues in distinct, standard rotamers is associated with a small shift in the helix register. At the C terminus, the helices splay apart and interact with a symmetry-related dimer in the crystal to form a short, anti-parallel, four-helix bundle. N-terminal fraying and C-terminal splaying of the helices, as well as the asymmetry and helix register shift describe unprecedented dynamic excursions of coiled coils. The low stability of APC-55 and divergence from the expected coiled-coil fold support the suggestion that the APC dimerization domain may extend beyond the first 55 residues.     

Structure of the N-terminal domain of the FOP (FGFR1OP) protein and implications for its dimerization and centrosomal localization

The fibroblast growth factor receptor 1 (FGFR1) oncogene partner, FOP, is a centrosomal protein that is involved in the anchoring of microtubules (MTS) to subcellular structures. The protein was originally discovered as a fusion partner with FGFR1 in oncoproteins that give rise to stem cell myeloproliferative disorders. A subsequent proteomics screen identified FOP as a component of the centrosome. FOP contains a Lis-homology (LisH) motif found in more than 100 eukaryotic proteins. LisH motifs are believed to be involved in microtubule dynamics and organization, cell migration, and chromosome segregation; several of them are associated with genetic diseases. We report here a 1.6A resolution crystal structure of the N-terminal dimerization domain of FOP. The structure comprises an alpha-helical bundle composed of two antiparallel chains, each of them having five alpha-helices. The central part of the dimer contains the LisH domain. We further determined that the FOP LisH domain is part of a longer N-terminal segment that is required, albeit not sufficient, for dimerization and centrosomal localization of FOP.     

Structure of the Toll/interleukin 1 receptor (TIR) domain of the immunosuppressive Brucella effector BtpA/Btp1/TcpB

BtpA/Btp1/TcpB is a virulence factor produced by Brucella species that possesses a Toll interleukin-1 receptor (TIR) domain. Once delivered into the host cell, BtpA interacts with MyD88 to interfere with TLR signalling and modulates microtubule dynamics. Here the crystal structure of the BtpA TIR domain at 3.15 Å is presented. The structure shows a dimeric arrangement of a canonical TIR domain, similar to the Paracoccus denitrificans Tir protein but secured by a unique long N-terminal α-tail that packs against the TIR:TIR dimer. Structure-based mutations and multi-angle light scattering experiments characterized the BtpA dimer conformation in solution. The structure of BtpA will help with studies to understand the mechanisms involved in its interactions with MyD88 and with microtubules.     

Solution structure of the E.coli TolA C-terminal domain reveals conformational changes upon binding to the phage g3p N-terminal domain

The Tol-Pal system of Escherichia coli is a macromolecular complex located in the cell envelope. It is involved in maintaining the integrity of the outer membrane and is required for the uptake of two different types of macromolecules, which are bacteriotoxins (colicins) and DNA of filamentous bacteriophages. The TolA protein plays a central role in these import mechanisms. Its C-terminal domain (TolAIII) is involved in the translocation step via direct interaction with the N-terminal domain of colicins and the N-terminal domain of the phage minor coat gene 3 protein (g3pN1). Extreme behaviours of TolAIII have been previously observed, since the structure of TolAIII either remained unaffected or adopted disordered conformation upon binding to different pore-forming colicins. Here, we have solved the 3D structure of free TolAIII by heteronuclear NMR spectroscopy and compared it to the crystal structure of TolAIII bound to g3pN1 in order to study the effect of g3pN1 on the tertiary structure of TolAIII. Backbone 1H, 15N and 13C resonances of the g3pN1-bound TolAIII were also assigned and used to superimpose the solution structure of free TolAIII on the crystal structure of the g3pN1-TolAIII fusion protein. This allowed us to track conformational changes of TolAIII upon binding. While the global fold of free TolAIII is mainly identical to that of g3pN1-bound TolAIII, shift of secondary structures does occur. Thus, TolAIII, which interacts also in vivo with Pal and TolB, is able to adapt its conformation upon binding to various partners. Possible models for protein binding mechanisms are discussed to explain this so-far unobserved behaviour of TolAIII.     

Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.     

Kar3Vik1 Mechanochemistry Is Inhibited by Mutation or Deletion of the C Terminus of the Vik1 Subunit

Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.