José M. Valpuesta

Polymerization of τ into Filaments in the Presence of Heparin: The Minimal Sequence Required for τ ‐ τ Interaction

Abstract: Paired helical filaments isolated from the brains of patients with Alzheimers disease are composed of a major protein component, the microtubule‐associated protein termed τ, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of τ into filamentous structures, we have analyzed the ability of the whole τ protein or some of its fragments to self‐assemble in the presence of heparin. Different τ fragments, all of them containing some sequences of the tubulin‐binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18‐residue‐long peptide corresponding to the third tubulin‐binding motif of τ. This suggests that the ability of τ for self‐assembly could be localized in a short sequence of amino acids present in the tubulin‐binding repeats of the τ molecule.

Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response

A long-standing paradox in the study of T cell antigen recognition is that of the high specificity–low affinity T cell receptor (TCR)–major histocompatibility complex peptide (MHCp) interaction. The existence of multivalent TCRs could resolve this paradox because they can simultaneously improve the avidity observed for monovalent interactions and allow for cooperative effects. We have studied the stoichiometry of the TCR by Blue Native–polyacrylamide gel electrophoresis and found that the TCR exists as a mixture of monovalent (αβγɛδɛζζ) and multivalent complexes with two or more ligand-binding TCRα/β subunits. The coexistence of monovalent and multivalent complexes was confirmed by electron microscopy after label fracture of intact T cells, thus ruling out any possible artifact caused by detergent solubilization. We found that although only the multivalent complexes become phosphorylated at low antigen doses, both multivalent and monovalent TCRs are phosphorylated at higher doses. Thus, the multivalent TCRs could be responsible for sensing low concentrations of antigen, whereas the monovalent TCRs could be responsible for dose-response effects at high concentrations, conditions in which the multivalent TCRs are saturated. Thus, besides resolving TCR stoichiometry, these data can explain how T cells respond to a wide range of MHCp concentrations while maintaining high sensitivity.

Eukaryotic type II chaperonin CCT interacts with actin through specific subunits.

Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP–1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and α-actin by cryo-electron microscopy and image processing. This shows that α-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT–substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTδ and the large domain to CCTβ or CCTε (both in position 1,4 with respect to δ). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.

Structure and function of a protein folding machine: the eukaryotic cytosolic chaperonin CCT

Chaperonins are large oligomers made up of two superimposed rings, each enclosing a cavity used for the folding of other proteins. Among the chaperonins, the eukaryotic cytosolic chaperonin CCT is the most complex, not only with regard to its subunit composition but also with respect to its function, still not well understood. Unlike the more well studied eubacterial chaperonin GroEL, which binds any protein that presents stretches of hydrophobic residues, CCT recognises in its substrates specific binding determinants and interacts with them through particular combinations of CCT subunits. Folding then occurs after the conformational changes induced in the chaperonin upon nucleotide binding have occurred, through a mechanism that, although still poorly defined, clearly differs from the one established for GroEL. Although CCT seems to be mainly involved in the folding of actin and tubulin, other substrates involved in various cellular roles are beginning to be characterised, including many WD40‐repeat, 7‐blade propeller proteins.

Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations

Three‐dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the α and β isoforms) using five specific CCT subunits. The CCT–tubulin interaction has a different geometry to the CCT–actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT‐bound conformation suggests a common mode of chaperonin–substrate interaction. CCT stabilizes quasi‐native structures in both proteins that are open through their domain‐connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding.

Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP‐1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD‐bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three‐dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD.

Structure of viral connectors and their function in bacteriophage assembly and DNA packaging

The viruses have been an attractive model for the study of basic mechanisms of protein/protein and protein/nucleic acid interactions involved in the assembly of macromolecular aggregates. This has been due primarily to their relative genetic simplicity as compared to their structural and functional complexity. Although most of the initial studies were carried out on bacterial and plant viruses, increasing data has also been accumulated from animal viruses, which has led to an understanding of some basic principles, as well as to many specific strategies in every system. The study of virus assembly has been a source of ideas that underlie our present knowledge of the organization of biological systems. It has also provided, since the production of bacteriophage mutants which have allowed the study of assembly intermediates, the first system in which the genetic studies played a dominant role. The increasing volume of data over the last years has revealed how the structural components can interact sequentially through an ordered pathway to yield macromolecular assemblies that satisfy the demands of stability required for a successful transfer of genetic information from host to host.

The structure of a biologically active influenza virus ribonucleoprotein complex

The influenza viruses contain a segmented, single-stranded RNA genome of negative polarity. Each RNA segment is encapsidated by the nucleoprotein and the polymerase complex into ribonucleoprotein particles (RNPs), which are responsible for virus transcription and replication. Despite their importance, information about the structure of these RNPs is scarce. We have determined the three-dimensional structure of a biologically active recombinant RNP by cryo-electron microscopy. The structure shows a nonameric nucleoprotein ring (at 12 Å resolution) with two monomers connected to the polymerase complex (at 18 Å resolution). Docking the atomic structures of the nucleoprotein and polymerase domains, as well as mutational analyses, has allowed us to define the interactions between the functional elements of the RNP and to propose the location of the viral RNA. Our results provide the first model for a functional negative-stranded RNA virus ribonucleoprotein complex. The structure reported here will serve as a framework to generate a quasi-atomic model of the molecular machine responsible for viral RNA synthesis and to test new models for virus RNA replication and transcription.

The Structure of Native Influenza Virion Ribonucleoproteins

Influenza Revealed Influenza virus, a single-stranded RNA virus, is responsible for substantial morbidity and mortality worldwide. The influenza ribonucleoprotein (RNP) complex, which carries out viral replication and transcription, is central to the virus life-cycle and to viral host adaptation (see the Perspective by Tao and Zheng). Structural characterization of the viral RNP has been challenging, but Moeller et al. (p. 1631, published online 22 November) and Arranz et al. (p. 1634, published online 22 November) now report the structure and assembly of this complex, using cryo-electron microscopy and negative-stain electron microscopy. The structures reveal how the viral polymerase, RNA genome, and nucleoprotein interact in the RNP providing insight into mechanisms for influenza genome replication and transcription. Electron microscopic analysis of a purified RNA-protein complex links its structure to the influenza life cycle. The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. The segmented negative-stranded RNAs are associated with the polymerase complex and nucleoprotein (NP), forming ribonucleoproteins (RNPs), which are responsible for virus transcription and replication. We describe the structure of native RNPs derived from virions. They show a double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end. This structure will be relevant for unraveling the mechanisms of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.

Structure of the Hsp110:Hsc70 Nucleotide Exchange Machine

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner proteins NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.