A smooth and differentiable bulk-solvent model for macromolecular diffraction.

Inclusion of low-resolution data in macromolecular crystallography requires a model for the bulk solvent. Previous methods have used a binary mask to accomplish this, which has proven to be very effective, but the mask is discontinuous at the solute-solvent boundary (i.e. the mask value jumps from zero to one) and is not differentiable with respect to atomic parameters. Here, two algorithms are introduced for computing bulk-solvent models using either a polynomial switch or a smoothly thresholded product of Gaussians, and both models are shown to be efficient and differentiable with respect to atomic coordinates. These alternative bulk-solvent models offer algorithmic improvements, while showing similar agreement of the model with the observed amplitudes relative to the binary model as monitored using R, R(free) and differences between experimental and model phases. As with the standard solvent models, the alternative models improve the agreement primarily with lower resolution (>6 A) data versus no bulk solvent. The models are easily implemented into crystallographic software packages and can be used as a general method for bulk-solvent correction in macromolecular crystallography.

Highly specific interactions between botulinum neurotoxins and synaptic vesicle proteins.

Despite its extreme toxicity, botulinum neurotoxin is widely utilized in low doses as a treatment for several neurological disorders; higher doses cause the neuroparalytic syndrome botulism. The toxin blocks neurotransmitter release by preferentially attaching to pre-synaptic membrane receptors at neuromuscular junctions and subsequently delivering a Zn2+-dependent protease component to presynaptic neuronal cytosol. These highly specialized enzymes exclusively hydrolyze peptide bonds within SNARE (soluble N-ethylmaleiamide sensitive factor attachment protein receptor) proteins. In this review we discuss the structural basis for botulinum toxin's exquisite specificity for its neuronal cell-surface receptors and intracellular SNARE targets.

Structure of proteins involved in synaptic vesicle fusion in neurons.

The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Structures of the SNARE complex, synaptotagmin III, nSec1, domains of the NSF chaperone and its adaptor SNAP, and Rab3 and some of its effectors provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models.

Structural insights into the molecular mechanism of calcium-dependent vesicle-membrane fusion.

The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Recently determined structures of the SNARE complex, synaptotagmin III, nSec1, domains of the NSF chaperone and its adaptor (SNAP), and Rab3 and some of its effectors provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models. Ultimately, knowledge of the structures of higher-order complexes and their dynamic behavior will be required to obtain a full understanding of the vesicle fusion protein machinery.

Polar residues drive association of polyleucine transmembrane helices.

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161-166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154-160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Structural characterization of the intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95.

PSD-95/SAP90 is a member of the MAGUK superfamily. In excitatory synapses, PSD-95 clusters receptors and ion channels at specific sites in the postsynaptic membrane and organizes downstream signaling and cytoskeletal molecules. We have determined the crystal structures of the apo and GMP-bound forms to 2.3 and 2.0 A resolutions, respectively, of a fragment containing the SH3, HOOK, and guanylate kinase (GK) domains of PSD-95. We observe an intramolecular interaction between the SH3 and GK domains involving the formation of a beta sheet including residues N- and C-terminal to the GK domain. Based on amino acid conservation and mutational data available in the literature, we propose that this intramolecular interaction is a common feature among MAGUK proteins.

Crystal structures of a Rab protein in its inactive and active conformations.

We have determined crystal structures of Sec4, a member of the Rab family in the G protein superfamily, in two states: bound to GDP, and to a non-hydrolyzable GTP analog, guanosine-5'-(beta, gamma)-imidotriphosphate (GppNHp). This represents the first structure of a Rab protein bound to GDP. Sec4 in both states grossly resembles other G proteins bound to GDP and GppNHp. In Sec4-GppNHp, structural features common to active Rab proteins are observed. In Sec4-GDP, the switch I region is highly disordered and displaced relative to the switch I region of Ras-GDP. In two of the four molecules of Sec4-GDP in the asymmetric unit of the Sec4-GDP crystals, the switch II region adopts a conformation similar to that seen in the structure of the small G protein Ran bound to GDP. This allows residues threonine 76, glutamate 80, and arginine 81 of Sec4 to make contacts with other conserved residues and water molecules important for nucleotide binding. In the other two molecules in the asymmetric unit, these interactions do not take place. This structural variability in both the switch I and switch II regions of GDP-bound Sec4 provides a possible explanation for the high off-rate of GDP bound to Sec4, and suggests a mechanism for regulation of the GTPase cycle of Rab proteins by GDI proteins.

Single-wavelength anomalous diffraction phasing revisited.

Multiwavelength anomalous diffraction (MAD) phasing has become a routinely used tool for determining new macromolecular structures. The MAD method has stringent data-collection requirements, typically necessitating radiation-resistant crystals and access to a tunable synchrotron beamline. In cases where synchrotron time, monochromator tunability or radiation damage is a concern or where high-throughput structure determination is desired, phasing methods capable of producing interpretable electron-density maps from less data become attractive alternatives to MAD. The increasing availability of tunable synchrotron data-collection facilities prompted the authors to revisit single-wavelength anomalous diffraction (SAD) phasing used in conjunction with a phase-ambiguity resolving method such as solvent flattening. The anomalous diffraction from seven different selenomethionine-labelled protein crystals has been analysed and it is shown that in conjunction with solvent flattening, diffraction data from the peak anomalous wavelength alone can produce interpretable electron-density maps of comparable quality to those resulting from full MAD phasing. Single-wavelength anomalous diffraction (SAD) phasing can therefore be a time-efficient alternative to MAD. The data also show that radiation damage can have a significant effect on the quality of SAD/MAD diffraction data. These results may be useful in the design of optimal strategies for collection of the diffraction data.

The 1.0 A crystal structure of Ca(2+)-bound calmodulin: an analysis of disorder and implications for functionally relevant plasticity.

Calmodulin (CaM) is a highly conserved 17 kDa eukaryotic protein that can bind specifically to over 100 protein targets in response to a Ca(2+) signal. Ca(2+)-CaM requires a considerable degree of structural plasticity to accomplish this physiological role; however, the nature and extent of this plasticity remain poorly characterized. Here, we present the 1.0 A crystal structure of Paramecium tetraurelia Ca(2+)-CaM, including 36 discretely disordered residues and a fifth Ca(2+) that mediates a crystal contact. The 36 discretely disordered residues are located primarily in the central helix and the two hydrophobic binding pockets, and reveal correlated side-chain disorder that may assist target-specific deformation of the binding pockets. Evidence of domain displacements and discrete backbone disorder is provided by translation-libration-screw (TLS) analysis and multiconformer models of protein disorder, respectively. In total, the evidence for disorder at every accessible length-scale in Ca(2+)-CaM suggests that the protein occupies a large number of hierarchically arranged conformational substates in the crystalline environment and may sample a quasi-continuous spectrum of conformations in solution. Therefore, we propose that the functionally distinct forms of CaM are less structurally distinct than previously believed, and that the different activities of CaM in response to Ca(2+) may result primarily from Ca(2+)-mediated alterations in the dynamics of the protein.

Structural insights into the molecular mechanism of Ca(2+)-dependent exocytosis.

The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Recent structures of the SNARE complex, synaptotagmin III, nSec1, domains of NSF and its adaptor SNAP, along with Rab3 and some of its effectors, provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models. Ultimately, this knowledge of the structures of higher-order complexes and their dynamic behavior will allow us to obtain a full understanding of the vesicle fusion protein machinery.

Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF).

Epsin (Eps15 interactor) is a cytosolic protein involved in clathrin-mediated endocytosis via its direct interactions with clathrin, the clathrin adaptor AP-2, and Eps15. The NH(2)-terminal portion of epsin contains a phylogenetically conserved module of unknown function, known as the ENTH domain (epsin NH(2)-terminal homology domain). We have now solved the crystal structure of rat epsin 1 ENTH domain to 1.8 A resolution. This domain is structurally similar to armadillo and Heat repeats of beta-catenin and karyopherin-beta, respectively. We have also identified and characterized the interaction of epsin 1, via the ENTH domain, with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). Leptomycin B, an antifungal antibiotic, which inhibits the Crm1- dependent nuclear export pathway, induces an accumulation of epsin 1 in the nucleus. These findings suggest that epsin 1 may function in a signaling pathway connecting the endocytic machinery to the regulation of nuclear function.

Interhelical hydrogen bonding drives strong interactions in membrane proteins.

Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins. To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper. We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence. Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur. Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.

Dramatic structural and thermodynamic consequences of repacking a protein's hydrophobic core.

BACKGROUND: Rop is an RNA binding, dimeric, four-helix bundle protein with a well-defined, regular hydrophobic core ideally suited for redesign studies. A family of Rop variants in which the hydrophobic core was systematically redesigned has previously been created and characterized. RESULTS: We present a structural and thermodynamic analysis of Ala2Ile2-6, a variant of Rop with an extensively redesigned hydrophobic core. The structure of Ala2Ile2-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface. The free-energy profile of Ala2Ile2-6 is also very different from that of wild-type Rop. Ala2Ile2-6 has a higher melting temperature than Rop, but undergoes a slightly smaller free-energy change on unfolding. CONCLUSIONS: The structure of Ala2Ile2-6, along with molecular modeling results, demonstrate the importance of tight packing of core residues and the adoption of favorable core side chain rotamer values in determining helix-helix interactions in the four-helix bundle fold. Structural disorder at the N and C termini of Ala2Ile2-6 provides a basis for the large differences in the enthalpy and entropy of Ala2Ile2-6 folding compared with wildtype Rop.

Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction.

Synaptotagmins are synaptic vesicle-associated, phospholipid-binding proteins most commonly associated with Ca(+2)-dependent exocytotic and Ca(+2)- independent endocytotic events. Synaptotagmin III is a 63.2-kD member of the synaptotagmin homology group; one of its characteristic properties is the ability to bind divalent cations and accessory proteins promiscuously. In the cytosolic portion of this protein, a flexible seven-amino acid linker joins two homologous C2 domains. The C2A domain binds to phospholipid membranes and other accessory proteins in a divalent cation-dependent fashion. The C2B domain promotes binding to other C2B domains, as well as accessory proteins independent of divalent cations. The 3.2 A crystal structure of synaptotagmin III, residues 295-566, which includes the C2A and C2B domains, exhibits differences in the shape of the Ca(+2)-binding pocket, the electrostatic surface potential, and the stoichiometry of bound divalent cations for the two domains. These observations may explain the disparate binding properties of the two domains. The C2A and the C2B domains do not interact; synaptotagmin, therefore, covalently links two independent C2 domains, each with potentially different binding partners. A model of synaptotagmin's involvement in Ca(+2)-dependent regulation of membrane fusion through its interaction with the SNARE complex is presented.

A highly automated heavy-atom search procedure for macromolecular structures.

A new highly automated heavy-atom search procedure combines a fast Fourier transform translation function, Patterson superposition functions and Patterson correlation refinement. The search procedure can be applied to various native and difference Patterson maps and their statistically weighted averages. The procedure was tested with diffraction data for several crystal structures with up to 30 heavy-atom sites in the asymmetric unit and with minimum Bragg spacings ranging from 3 to 4 A. In all cases, the correct sites were found with modest computing time.

Crystal structure of the vesicular transport protein Sec17: implications for SNAP function in SNARE complex disassembly.

SNAP proteins play an essential role in membrane trafficking in eukaryotic cells. They activate and recycle SNARE proteins by serving as adaptors between SNAREs and the cytosolic chaperone NSF. We have determined the crystal structure of Sec17, the yeast homolog of alpha-SNAP, to 2.9 A resolution. Sec17 is composed of an N-terminal twisted sheet of alpha-helical hairpins and a C-terminal alpha-helical bundle. The N-terminal sheet has local similarity to the tetratricopeptide repeats from protein phosphatase 5 but has a different overall twist. Sec17 also shares structural features with HEAT and clathrin heavy chain repeats. Possible models of SNAP:SNARE binding suggest that SNAPs may function as lever arms, transmitting forces generated by conformational changes in NSF/Sec18 to drive disassembly of SNARE complexes.

NSF N-terminal domain crystal structure: models of NSF function.

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase essential for eukaryotic vesicle fusion. Along with SNAP proteins, it disassembles cis-SNARE complexes upon ATP hydrolysis, preparing SNAREs for trans complex formation. We have determined the crystal structure of the N-terminal domain of NSF (N) to 1.9 A resolution. N contains two subdomains which form a groove that is a likely SNAP interaction site. Unexpectedly, both N subdomains are structurally similar to domains in EF-Tu. Based on this similarity, we propose a model for a large conformational change in NSF that drives SNARE complex disassembly.

Extending the limits of molecular replacement through combined simulated annealing and maximum-likelihood refinement.

Phases determined by the molecular-replacement method often suffer from model bias. In extreme cases, the refinement of the atomic model can stall at high free R values when the resulting electron-density maps provide little indication of how to correct the model, sometimes rendering even a correct solution unusable. Here, it is shown that several recent advances in refinement methodology allow productive refinement, even in cases where the molecular-replacement-phased electron-density maps do not allow manual rebuilding. In test calculations performed with a series of homologous models of penicillopepsin using either backbone atoms, or backbone atoms plus conserved core residues, model bias is reduced and refinement can proceed efficiently, even if the initial model is far from the correct one. These new methods combine cross-validation, torsion-angle dynamics simulated annealing and maximum-likelihood target functions. It is also shown that the free R value is an excellent indicator of model quality after refinement, potentially discriminating between correct and incorrect molecular-replacement solutions. The use of phase information, even in the form of bimodal single-isomorphous-replacement phase distributions, greatly improves the radius of convergence of refinement and hence the quality of the electron-density maps, further extending the limits of molecular replacement.