Claire Bagnéris

Crystal Structures of α-Crystallin Domain Dimers of αB-Crystallin and Hsp20

Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised alpha-crystallin domain from rat Hsp20 and that from human alphaB-crystallin show that they form homodimers with a shared groove at the interface by extending a beta sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the alphaB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G alpha-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.

Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing

Sodium-gated ion channels open and close in response to the flow of ions. Here, McCusker et al. report the open structure of a sodium-gated ion channel pore from a bacterial homologue, and show, by comparison with the closed structure, that the movement of a C-terminal helix is sufficient to open the channel.

The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes

Resuscitation-promoting factor (RPF) proteins reactivate stationary-phase cultures of (G+C)-rich Gram-positive bacteria including the causative agent of tuberculosis, Mycobacterium tuberculosis. We report the solution structure of the RPF domain from M. tuberculosis Rv1009 (RpfB) solved by heteronuclear multidimensional NMR. Structural homology with various glycoside hydrolases suggested that RpfB cleaved oligosaccharides. Biochemical studies indicate that a conserved active site glutamate is important for resuscitation activity. These data, as well as the presence of a clear binding pocket for a large molecule, indicate that oligosaccharide cleavage is probably the signal for revival from dormancy.

Crystal Structure of a vFlip-IKKγ Complex: Insights into Viral Activation of the IKK Signalosome

Key to the pathogenicity of several viruses is activation of the canonical nuclear factor-kappaB (NF-kappaB) transcriptional pathway. Subversion of this tightly regulated mechanism is achieved through the production of host mimetic viral proteins that deregulate the transcription process. One such protein is ks-vFLIP (produced by the Kaposis sarcoma herpes virus [KSHV]), which associates with IKKgamma, an essential component of the IKK complex or signalosome. This interaction renders the canonical NF-kappaB pathway constitutively active and has been linked to Kaposis sarcoma and other malignancies. In order to elucidate the molecular basis underpinning ks-vFLIP-induced activation of the IKK signalosome, we have determined the crystal structure of a complex involving a fragment of IKKgamma bound to ks-vFLIP at 3.2 A. In addition to identifying and subsequently probing the ks-vFLIP-IKKgamma interface, we have also investigated the effects of a mutation implicated in the genetic disorder anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID).

Molecular dynamics of ion transport through the open conformation of a bacterial voltage-gated sodium channel

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel’s full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.

Crystal structure of R120G disease mutant of human αB-crystallin domain dimer shows closure of a groove.

Small heat shock proteins form large cytosolic assemblies from an “α-crystallin domain” (ACD) flanked by sequence extensions. Mutation of a conserved arginine in the ACD of several human small heat shock protein family members causes many common inherited diseases of the lens and neuromuscular system. The mutation R120G in αB-crystallin causes myopathy, cardiomyopathy and cataract. We have solved the X-ray structure of the excised ACD dimer of human αB R120G close to physiological pH and compared it with several recently determined wild-type vertebrate ACD dimer structures. Wild-type excised ACD dimers have a deep groove at the interface floored by a flat extended “bottom sheet.” Solid-state NMR studies of large assemblies of full-length αB-crystallin have shown that the groove is blocked in the ACD dimer by curvature of the bottom sheet. The crystal structure of R120G ACD dimer also reveals a closed groove, but here the bottom sheet is flat. Loss of Arg120 results in rearrangement of an extensive array of charged interactions across this interface. His83 and Asp80 on movable arches on either side of the interface close the groove by forming two new salt bridges. The residues involved in this extended set of ionic interactions are conserved in Hsp27, Hsp20, αA- and αB-crystallin sequences. They are not conserved in Hsp22, where mutation of the equivalent of Arg120 causes neuropathy. We speculate that the αB R120G mutation disturbs oligomer dynamics, causing the growth of large soluble oligomers that are toxic to cells by blocking essential processes.

Prokaryotic Navms Channel as a Structural and Functional Model for Eukaryotic Sodium Channel Antagonism.

Significance Many drugs used to treat pain, epilepsy, and cardiac arrhythmias target human voltage-gated sodium-selective channels. Surprisingly, we found that a bacterial voltage-gated sodium channel is also inhibited by many eukaryotic sodium channel antagonists. This bacterial channel was crystallized with several brominated blocker compounds, and the high-resolution structures reveal a common antagonist binding site in the cavity of the pore. Electrophysiology studies of channels with mutations at adjacent residues validate the site. These results suggest that despite millions of years of evolution separating human and bacterial sodium channels, these simple bacterial channels can be a valuable tool for screening and rational design of human drugs. Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus.

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of time scales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here, we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations that regulate the interconversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour time scales and secondary to quaternary structural levels, and provides an approach for, obtaining simultaneously detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly.

Molecular structure of human geminin

The origin licensing repressor geminin is a unique bifunctional protein providing a molecular link between cellular proliferation, differentiation and genomic stability. Here we report the first molecular structure of human geminin, determined by EM and image processing at a resolution of 17.5 Å. The geminin molecule is a tetramer formed by two dimers with monomers interacting via coiled-coil domains. The unusual structural organization of geminin provides molecular insight into its bifunctional nature.

Structural model of the open–closed–inactivated cycle of prokaryotic voltage-gated sodium channels

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels.