Raimond B. G. Ravelli

Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain.

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 Å resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin–colchicine complex sheds light on the mechanism of colchicines activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.

Structural basis for the regulation of tubulin by vinblastine

Vinblastine is one of several tubulin-targeting Vinca alkaloids that have been responsible for many chemotherapeutic successes since their introduction in the clinic as antitumour drugs. In contrast with the two other classes of small tubulin-binding molecules (Taxol and colchicine), the binding site of vinblastine is largely unknown and the molecular mechanism of this drug has remained elusive. Here we report the X-ray structure of vinblastine bound to tubulin in a complex with the RB3 protein stathmin-like domain (RB3-SLD). Vinblastine introduces a wedge at the interface of two tubulin molecules and thus interferes with tubulin assembly. Together with electron microscopical and biochemical data, the structure explains vinblastine-induced tubulin self-association into spiral aggregates at the expense of microtubule growth. It also shows that vinblastine and the amino-terminal part of RB3-SLD binding sites share a hydrophobic groove on the α-tubulin surface that is located at an intermolecular contact in microtubules. This is an attractive target for drugs designed to perturb microtubule dynamics by interfacial interference, for which tubulin seems ideally suited because of its propensity to self-associate.

Adhesion mechanism of human beta 2-glycoprotein I to phospholipids based on its crystal structure

Human β2‐glycoprotein I is a heavily glycosylated five‐domain plasma membrane‐adhesion protein, which has been implicated in blood coagulation and clearance of apoptotic bodies from the circulation. It is also the key antigen in the autoimmune disease anti‐phospholipid syndrome. The crystal structure of β2‐glycoprotein I isolated from human plasma reveals an elongated fish‐hook‐like arrangement of the globular short consensus repeat domains. Half of the C‐terminal fifth domain deviates strongly from the standard fold, as observed in domains one to four. This aberrant half forms a specific phospholipid‐binding site. A large patch of 14 positively charged residues provides electrostatic interactions with anionic phospholipid headgroups and an exposed membrane‐insertion loop yields specificity for lipid layers. The observed spatial arrangement of the five domains suggests a functional partitioning of protein adhesion and membrane adhesion over the N‐ and C‐terminal domains, respectively, separated by glycosylated bridging domains. Coordinates are in the Protein Data Bank (accession No. 1QUB).

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

Structural changes occur in the αβ-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a β-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with α-tubulin from stacking onto a β-tubulin β sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the β-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.

Specific Radiation-Damage Can Be Used To Solve Macromolecular Crystal Structures

The use of third generation synchrotron sources has led to renewed concern about the effect of ionizing radiation on crystalline biological samples. In general, the problem is seen as one to be avoided. However, in this paper, it is shown that, far from being a hindrance to successful structure determination, radiation damage provides an opportunity for phasing macromolecular structures. This is successfully demonstrated for both a protein and an oligonucleotide, by way of which complete models were built automatically. The possibility that, through the exploitation of radiation damage, the phase problem could become less of a barrier to macromolecular crystal structure determination is discussed.

Structural Insights Into the Slit-Robo Complex.

Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1–2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 Å resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.

MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments

MxCuBE is a beamline control environment optimized for the needs of macromolecular crystallography. This paper describes the design of the software and the features that MxCuBE currently provides.

The 1.8-A Crystal Structure of α1-Acid Glycoprotein (Orosomucoid) Solved by UV RIP Reveals the Broad Drug-Binding Activity of This Human Plasma Lipocalin

Alpha(1)-acid glycoprotein (AGP) is an important drug-binding protein in human plasma and, as an acute-phase protein, it has a strong influence on pharmacokinetics and pharmacodynamics of many pharmaceuticals. We report the crystal structure of the recombinant unglycosylated human AGP at 1.8 A resolution, which was solved using the new method of UV-radiation-damage-induced phasing (UV RIP). AGP reveals a typical lipocalin fold comprising an eight-stranded beta-barrel. Of the four loops that form the entrance to the ligand-binding site, loop 1, which connects beta-strands A and B, is among the longest observed so far and exhibits two full turns of an alpha-helix. Furthermore, it carries one of the five N-linked glycosylation sites, while a second one occurs underneath the tip of loop 2. The branched, partly hydrophobic, and partly acidic cavity, together with the presumably flexible loop 1 and the two sugar side chains at its entrance, explains the diverse ligand spectrum of AGP, which is known to vary with changes in glycosylation pattern.

Fibril structure of amyloid-beta (1-42) by cryo-electron microscopy.

Elucidating pathological fibril structure Amyloid-β (Aβ) is a key pathological contributor to Alzheimers disease. Gremer et al. used cryoelectron microscopy data to build a high-quality de novo atomic model of Aβ fibrils (see the Perspective by Pospich and Raunser). The complete structure reveals all 42 amino acids (including the entire N terminus) and provides a structural basis for understanding the effect of several disease-causing and disease-preventing mutations. The fibril consists of two intertwined protofilaments with an unexpected dimer interface that is different from those proposed previously. The structure has implications for the mechanism of fibril growth and will be an important stepping stone to rational drug design. Science, this issue p. 116; see also p. 45 Cryo–electron microscopy structure of an amyloid-β(1–42) fibril reveals a protofilament interface and the entire N-terminal region. Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-β protein (Aβ) are the main component of the senile plaques found in brains of Alzheimer’s disease patients. We present the structure of an Aβ(1–42) fibril composed of two intertwined protofilaments determined by cryo–electron microscopy (cryo-EM) to 4.0-angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-β structure resulting in an overall “LS”-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed “groove” and “ridge,” leading to different binding pathways on both fibril ends, which has implications for fibril growth.