Detection and selective dissociation of intact ribosomes in a mass spectrometer.

Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg(2+) concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 +/- 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.

Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120).

The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor.

Structural basis for dimerization of the Dictyostelium gelation factor (ABP120) rod.

Gelation factor (ABP120) is one of the principal actin-cross-linking proteins of Dictyostelium discoideum. The extended molecule has an N-terminal 250-residue actin-binding domain and a rod constructed from six 100-residue repeats that have an Ig fold. The ability to dimerize is crucial to the actin cross-linking function of gelation factor and is mediated by the rod in which the two chains are arranged in an antiparallel fashion. We report the 2.2 A resolution crystal structure of rod domains 5 and 6, which shows that dimerization is mediated primarily by rod domain 6 and is the result of a double edge-to-edge extension of beta-sheets. Thus, contrary to earlier proposals, the chains of the dimeric gelation factor molecule overlap only within domain 6, and domains 1-5 do not pair with domains from the other chain. This information allows construction of a model of the gelation factor molecule and suggests how the chains in the related molecule filamin (ABP280) may interact.

Location of the binding site of the mannose-specific lectin comitin on F-actin.

We have used electron microscopy and computer image processing to produce a three-dimensional reconstruction of F-actin filaments decorated with the putative lectin and actin-binding protein comitin. These reconstructions show that comitin binds to F-actin at high radius primarily to actin subdomain 1. This location is distinctly different from the binding site on F-actin for other actin bundling proteins, such as members of the alpha-actinin family, and may result from the positively charged comitin interacting with negatively charged sites near the actin N terminus in subdomain 1. The location of the comitin binding site and its restriction to subdomain 1 on a single actin monomer is consistent with comitin's having a function distinct from other actin-binding proteins and, for example, would enable comitin to link bundled actin filaments to the Golgi.

The repeating segments of the F-actin cross-linking gelation factor (ABP-120) have an immunoglobulin-like fold.

The 120,000 M(r) gelation factor and alpha-actinin are among the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are rod-shaped homodimers. Each monomer chain is comprised of an actin-binding domain and a rod domain. The rod domain of the gelation factor consists of six 100-residue repetitive segments with high internal homology. We have now determined the three-dimensional structure of segment 4 of the rod domain of the gelation factor from D. discoideum using NMR spectroscopy. The segment consists of seven beta-sheets arranged in an immunoglobulin-like (Ig) fold. This is completely different from the alpha-actinin rod domain which consists of four spectrin-like alpha-helical segments. The gelation factor is the first example of an Ig-fold found in an actin-binding protein. Two highly homologous actin-binding proteins from human with similar sequences to the gelation factor, filamin and a 280,000 M(r) actin-binding protein (ABP-280), share conserved residues that form the core of the gelation factor repetitive segment structure. Thus, the segment 4 structure should be common to this subfamily of the spectrin superfamily. The structure of segment 4 together with previously published electron microscopy data, provide an explanation for the dimerization of the whole gelation factor molecule.

Crystallization and preliminary X-Ray diffraction characterization of a dimerizing fragment of the rod domain of the Dictyostelium gelation factor (ABP-120).

We have expressed in Escherichia coli a construct corresponding to sequence repeats 5 and 6 of the rod domain of the actin-binding protein Dictyostelium gelation factor (ABP-120). We have obtained orthorhombic P212121 crystals of the protein with a = 43.5 A, b = 103.2 A, c = 124.4 A. These crystals diffract past 2.2 A resolution using synchrotron radiation and are suitable for high-resolution structural analysis. ABP-120 is a key component of the Dictyostelium cytoskeleton, where it functions to crosslink F-actin filaments into networks. This crosslinking function of ABP-120 depends crucially on the formation of dimeric molecules that contain an actin-binding site on each chain, and this dimerization is brought about through interactions between repeating sequence modules in the rod domain. Because the construct we have expressed retains the ability to dimerize, it should enable us to establish the precise manner in which these sequence repeats interact with one another in the intact molecule.

Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity.

Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli. Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135. The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton.