Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.     

A solution SAXS study of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet.

The crystal structure of a soluble form of Borrelia burgdorferi outer surface protein A (OspA) complexed with the Fab fragment of a monoclonal antibody has revealed an unusual structure that has a repetitive antiparallel beta topology with a nonglobular, single layer beta-sheet connecting the globular N- and C-terminal domains. Earlier NMR studies have shown that the local structure of OspA including the single layer beta-sheet is similar to the crystal structure. Here we report a small angle X-ray scattering (SAXS) study of the global conformation of OspA in solution. The radius of gyration (Rg) and the length distribution function (P(r)) of OspA measured by SAXS in solution are nearly identical to the calculated ones from the crystal structure, respectively. The NMR and SAXS experiments complement each other to show that OspA including the central single-layer beta-sheet is a stable structure in solution, and that the OspA crystal structure represents the predominant solution conformation of the protein.     

Structural Insights into the Globular Tails of the Human Type V Myosins Myo5a,Myo5b,and Myo5c

Vertebrate type V myosins (MyoV) Myo5a, Myo5b, and Myo5c mediate transport of several different cargoes. All MyoV paralogs bind to cargo complexes mainly by their C-terminal globular domains. In absence of cargo, the globular domain of Myo5a inhibits its motor domain. Here, we report low-resolution SAXS models for the globular domains from human Myo5a, Myo5b, and Myo5c, which suggest very similar overall shapes of all three paralogs. We determined the crystal structures of globular domains from Myo5a and Myo5b, and provide a homology model for human Myo5c. When we docked the Myo5a crystal structure into a previously published electron microscopy density of the autoinhibited full-length Myo5a, only one domain orientation resulted in a good fit. This structural arrangement suggests the participation of additional region of the globular domain in autoinhibition. Quantification of the interaction of the Myo5a globular domain with its motor complex revealed a tight binding with dissociation half-life in the order of minutes, suggesting a rather slow transition between the active and inactive states.     

Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone.

The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 A. The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them. The structure has one bound zinc pentavalently coordinated to residues from both domains. Unlike previous PLBP structures that have an interdomain hinge composed of beta-strands, the N- and C-domains of TroA are linked by a single long backbone helix. This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange.     

Dengue virus nonstructural protein 5 adopts multiple conformations in solution

Dengue virus (DENV) nonstructural protein 5 (NS5) is composed of two globular domains separated by a 10-residue linker. The N-terminal domain participates in the synthesis of a mRNA cap 1 structure ((7Me)GpppA(2'OMe)) at the 5' end of the viral genome and possesses guanylyltransferase, guanine-N7-methyltransferase, and nucleoside-2'O-methyltransferase activities. The C-terminal domain is an RNA-dependent RNA polymerase responsible for viral RNA synthesis. Although crystal structures of the two isolated domains have been obtained, there are no structural data for full-length NS5. It is also unclear whether the two NS5 domains interact with each other to form a stable structure in which the relative orientation of the two domains is fixed. To investigate the structure and dynamics of DENV type 3 NS5 in solution, we conducted small-angle X-ray scattering experiments with the full-length protein. NS5 was found to be monomeric and well-folded under the conditions tested. The results of these experiments also suggest that NS5 adopts multiple conformations in solution, ranging from compact to more extended forms in which the two domains do not seem to interact with each other. We interpret the multiple conformations of NS5 observed in solution as resulting from weak interactions between the two NS5 domains and flexibility of the linker in the absence of other components of the replication complex.     

Structural dynamics of calmodulin and troponin C

We present the results of computational simulation studies of the structures of calmodulin (CAM) and troponin C (TNC). Possible differences between the structures of these molecules in the crystal and in solution were suggested by results from some recent experimental studies, which implied that their conformations in solution may be more compacted than the characteristic dumbbell shape observed in the crystal. The molecular dynamics simulations were carried out with the CHARMM system of programs, and the environment was modeled with a distance-dependent dielectric permittivity and discrete water molecules surrounding the proteins at starting positions identified in the crystals of CAM and TNC. Methods of macromolecular structure analysis, including linear distance plots, distance matrices and a matrix representation of hydrogen bonding, were used to analyze the nature, the extent and the source of structural differences between the computed structures of the molecules and their conformations in the crystal. Following the longest simulation, in which intradomain structure was conserved, the crystallographically observed dumbbell structure of the molecule changed due to a kinking or bending in the region of the central tether helix connecting the two Ca(2+)-binding domains which moved into close proximity. The resulting structure correlates with experimental observations of complexes between CAM and peptides such as melittin and mastoparan. Analysis of the corresponding pair distance distribution functions in comparison to experimental results suggests the dynamic existence of a non-negligible fraction of the compacted structure in aqueous solutions of CAM. In this more nearly globular shape, CAM reveals to the environment two interior pockets that contain a number of hydrophobic residues, in agreement with NMR data suggesting involvement of such residues in the binding of inhibitors and proteins to CAM.     

Solution structure of calmodulin and its complex with a myosin light chain kinase fragment.

The solution structure of Ca2+ ligated calmodulin and of its complex with a 26-residue peptide fragment of skeletal muscle myosin light chain kinase (skMLCK) have been investigated by multi-dimensional NMR. In the absence of peptide, the two globular domains of calmodulin adopt the same structure as observed in the crystalline form. The so-called 'central helix' which is observed in the crystalline state is disrupted in solution. 15N relaxation studies show that residues Asp78 through Ser81, located near the middle of this 'central helix', form a very flexible link between the two globular domains. In the presence of skMLCK target peptide, the peptide-protein complex adopts a globular ellipsoidal shape. The helical peptide is located in a hydrophobic channel that goes through the center of the complex and makes an angle of approximately 45 degrees with the long axis of the ellipsoid.     

What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.     

Integrated analysis of the conformation of a protein-linked spin label by crystallography,EPR and NMR spectroscopy

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.     

Structure and dynamics of calcium-activated calmodulin in solution.

Two 4-ns molecular dynamics simulations of calcium loaded calmodulin in solution have been performed, using both standard nonbonded cutoffs and Ewald summation to treat electrostatic interactions. Our simulation results are generally consistent with solution experimental studies of calmodulin structure and dynamics, including NMR, cross-linking, fluorescence and x-ray scattering. The most interesting result of the molecular dynamics simulations is the detection of large-scale structural fluctuations of calmodulin in solution. The globular N- and C-terminal domains tend to move approximately like rigid bodies, with fluctuations of interdomain distances within a 7 A range and of interdomain angles by up to 60 deg. Essential dynamics analysis indicates that the three dominant types of motion involve bending of the central helix in two perpendicular planes and a twist in which the domains rotate in opposite directions around the central helix. In the more realistic Ewald trajectory the protein backbone remains mostly within a 2-3 A root-mean-square distance from the crystal structure, the secondary structure within the domains is conserved and middle part of the central helix becomes disordered. The central helix itself exhibits limited fluctuations, with its bend angle exploring the 0-50 degrees range and the end-to-end distance falling in 39-43 A. The results of the two simulations were similar in many respects. However, the cutoff trajectory exhibited a larger deviation from the crystal, loss of several helical hydrogen bonds in the N-terminal domain and lack of structural disorder in the central helix.     

Crystal structure of the SMC head domain: an ABC ATPase with 900 residues antiparallel coiled-coil inserted

SMC (structural maintenance of chromosomes) proteins are large coiled-coil proteins involved in chromosome condensation, sister chromatid cohesion, and DNA double-strand break processing. They share a conserved five-domain architecture with three globular domains separated by two long coiled-coil segments. The coiled-coil segments are antiparallel, bringing the N and C-terminal globular domains together. We have expressed a fusion protein of the N and C-terminal globular domains of Thermotoga maritima SMC in Escherichia coli by replacing the approximately 900 residue coiled-coil and hinge segment with a short peptide linker. The SMC head domain (SMChd) binds and condenses DNA in an ATP-dependent manner. Using selenomethionine-substituted protein and multiple anomalous dispersion phasing, we have solved the crystal structure of the SMChd to 3.1 A resolution. In the monoclinic crystal form, six SMChd molecules form two turns of a helix. The fold of SMChd is closely related to the ATP-binding cassette (ABC) ATPase family of proteins and Rad50, a member of the SMC family involved in DNA double-strand break repair. In SMChd, the ABC ATPase fold is formed by the N and C-terminal domains with the 900 residue coiled-coil and hinge segment inserted in the middle of the fold. The crystal structure of an SMChd confirms that the coiled-coil segments in SMC proteins are anti-parallel and shows how the N and C-terminal domains come together to form an ABC ATPase. Comparison to the structure of the MukB N-terminal domain demonstrates the close relationship between MukB and SMC proteins, and indicates a helix to strand conversion when N and C-terminal parts come together.     

Crystal structure of a putative type I restriction-modification S subunit from Mycoplasma genitalium

The crystal structure of the eubacteria Mycoplasma genitalium ORF MG438 polypeptide, determined by multiple anomalous dispersion and refined at 2.3 A resolution, reveals the organization of S subunits from the Type I restriction and modification system. The structure consists of two globular domains, with about 150 residues each, separated by a pair of 40 residue long antiparallel alpha-helices. The globular domains correspond to the variable target recognition domains (TRDs), as previously defined for S subunits on sequence analysis, while the two helices correspond to the central (CR1) and C-terminal (CR2) conserved regions, respectively. The structure of the MG438 subunit presents an overall cyclic topology with an intramolecular 2-fold axis that superimposes the N and the C-half parts, each half containing a globular domain and a conserved helix. TRDs are found to be structurally related with the small domain of the Type II N6-adenine DNA MTase TaqI. These relationships together with the structural peculiarities of MG438, in particular the presence of the intramolecular quasi-symmetry, allow the proposal of a model for S subunits recognition of their DNA targets in agreement with previous experimental results. In the crystal, two subunits of MG438 related by a crystallographic 2-fold axis present a large contact area mainly involving the symmetric interactions of a cluster of exposed hydrophobic residues. Comparison with the recently reported structure of an S subunit from the archaea Methanococcus jannaschii highlights the structural features preserved despite a sequence identity below 20%, but also reveals important differences in the globular domains and in their disposition with respect to the conserved regions.     

NMR studies on the substrate-binding domains of the thermosome: structural plasticity in the protrusion region

Group II chaperonins close their cavity with the help of conserved, helical extensions, the so-called protrusions, which emanate from the apical or substrate-binding domains. A comparison of previously solved crystal structures of the apical domains of the thermosome from Thermoplasma acidophilum showed structural plasticity in the protrusion parts induced by extensive packing interactions. In order to assess the influence of the crystal contacts we investigated both the alpha and beta-apical domains (alpha-ADT and beta-ADT) in solution by NMR spectroscopy. Secondary structure assignments and 15N backbone relaxation measurements showed mostly rigid structural elements in the globular parts of the domains, but revealed intrinsic structural disorder and partial helix fraying in the protrusion regions. On the other hand, a beta-turn-motif conserved in archaeal group II chaperonins might facilitate substrate recognition. Our results help us to specify the idea of the open, substrate-accepting state of the thermosome and may provide an additional jigsaw piece in understanding the mode of substrate binding of group II chaperonins.     

Cooperative binding of the globular domains of histones H1 and H5 to DNA.

In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.     

Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.