Luke M. Rice

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Regulation of microtubule motors by tubulin isotypes and post-translational modifications

The ‘tubulin-code’ hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogeneous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 α/β-tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single-molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal β-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of α-tubulin. Our results also show that different molecular motors recognize distinctive tubulin ‘signatures’, which supports the premise of the tubulin-code hypothesis.

New applications of simulated annealing in X-ray crystallography and solution NMR.

The recent computational developments discussed in this review will become available on the internet (http://atb.csb.yale.edu) in the near future. We thank Temple Burling, Michael Nilges and Gregory Warren for critical reading of the manuscript. LMR is an HHMI predoctoral fellow. This work was funded in part by grants from the National Science Foundation to ATB (BIR 9514819 and ASC 93-181159).

Multiple Conformations of E. coli Hsp90 in Solution: Insights into the Conformational Dynamics of Hsp90

Hsp90, an essential eukaryotic chaperone, depends upon its intrinsic ATPase activity for function. Crystal structures of the bacterial Hsp90 homolog, HtpG, and the yeast Hsp90 reveal large domain rearrangements between the nucleotide-free and the nucleotide-bound forms. We used small-angle X-ray scattering and recently developed molecular modeling methods to characterize the solution structure of HtpG and demonstrate how it differs from known Hsp90 conformations. In addition to this HtpG conformation, we demonstrate that under physiologically relevant conditions, multiple conformations coexist in equilibrium. In solution, nucleotide-free HtpG adopts a more extended conformation than observed in the crystal, and upon the addition of AMPPNP, HtpG is in equilibrium between this open state and a closed state that is in good agreement with the yeast AMPPNP crystal structure. These studies provide a unique view of Hsp90 conformational dynamics and provide a model for the role of nucleotide in effecting conformational change.

Insights into microtubule nucleation from the crystal structure of human gamma-tubulin.

Microtubules are hollow polymers of αβ-tubulin that show GTP-dependent assembly dynamics and comprise a critical part of the eukaryotic cytoskeleton. Initiation of new microtubules in vivo requires γ-tubulin, organized as an oligomer within the 2.2-MDa γ-tubulin ring complex (γ-TuRC) of higher eukaryotes. Structural insight is lacking regarding γ-tubulin, its oligomerization and how it promotes microtubule assembly. Here we report the 2.7-Å crystal structure of human γ-tubulin bound to GTP-γS (a non-hydrolysable GTP analogue). We observe a ‘curved’ conformation for γ-tubulin–GTPγS, similar to that seen for GDP-bound, unpolymerized αβ-tubulin. Tubulins are thought to represent a distinct class of GTP-binding proteins, and conformational switching in γ-tubulin might differ from the nucleotide-dependent switching of signalling GTPases. A crystal packing interaction replicates the lateral contacts between α- and β-tubulins in the microtubule, and this association probably forms the basis for γ-tubulin oligomerization within the γ-TuRC. Laterally associated γ-tubulins in the γ-TuRC might promote microtubule nucleation by providing a template that enhances the intrinsically weak lateral interaction between αβ-tubulin heterodimers. Because they are dimeric, αβ-tubulins cannot form microtubule-like lateral associations in the curved conformation. The lateral array of γ-tubulins we observe in the crystal reveals a unique functional property of a monomeric tubulin.

The lattice as allosteric effector: structural studies of alphabeta- and gamma-tubulin clarify the role of GTP in microtubule assembly

GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, αβ-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in αβ-tubulin. The allosteric model posits that unpolymerized αβ-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of αβ-tubulin, the 2.3 Å crystal structure of γ-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous γ-tubulin:GTPγS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of αβ-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive αβ-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. αβ-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior.

Crystallographic refinement by simulated annealing: Methods and applications

Publisher Summary This chapter discusses crystallographic refinement, a technique aimed at optimizing the agreement of an atomic model with both observed diffraction data and chemical restraints. Optimization problems in macromolecular crystallography generally suffer from there being multiple minima, which arise largely from the high dimensionality of the parameter space. The many local minima of the target function tend to defeat gradient descent optimization techniques, such as conjugate gradient or least-squares methods. These methods are simply not capable of shifting the atomic coordinates enough to correct errors in the initial model. Simulated annealing has improved the efficiency of crystallographic refinement significantly. However, simulated annealing refinement alone is still insufficient to refine a crystal structure automatically without human intervention. With currently available computing power, tedious manual adjustments, using computer graphics to display and move positions of atoms of the model in the electron-density maps, can represent the rate-limiting step in the refinement process.

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.

Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP- 25 homolog, Sec9

SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells. The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, andsoluble N -ethylmaleimide-sensitivefactor attachment protein (α-SNAP), have been extensively studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast α-SNAP homolog, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion. In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo. As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.

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.