The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein–ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community

Regulator of G-Protein Signaling 14 (RGS14) Is a Selective H-Ras Effector

Background: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Ga-mediated GTP hydrolysis (‘‘GTPase-accelerating proteins’’ or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Ga GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras.Methodology/Principal Findings: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co- transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor- mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling.Conclusions/Significance: In cells, RGS14 facilitates the formation of a selective Ras?GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras- binding domain architecture with RGS14

Subcortical brain volume, regional cortical thickness, and cortical surface area across disorders: findings from the ENIGMA ADHD, ASD, and OCD Working Groups

Objective Attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and obsessive-compulsive disorder (OCD) are common neurodevelopmental disorders that frequently co-occur. We aimed to directly compare all three disorders. The ENIGMA consortium is ideally positioned to investigate structural brain alterations across these disorders. Methods Structural T1-weighted whole-brain MRI of controls (n=5,827) and patients with ADHD (n=2,271), ASD (n=1,777), and OCD (n=2,323) from 151 cohorts worldwide were analyzed using standardized processing protocols. We examined subcortical volume, cortical thickness and surface area differences within a mega-analytical framework, pooling measures extracted from each cohort. Analyses were performed separately for children, adolescents, and adults using linear mixed-effects models adjusting for age, sex and site (and ICV for subcortical and surface area measures). Results We found no shared alterations among all three disorders, while shared alterations between any two disorders did not survive multiple comparisons correction. Children with ADHD compared to those with OCD had smaller hippocampal volumes, possibly influenced by IQ. Children and adolescents with ADHD also had smaller ICV than controls and those with OCD or ASD. Adults with ASD showed thicker frontal cortices compared to adult controls and other clinical groups. No OCD-specific alterations across different age-groups and surface area alterations among all disorders in childhood and adulthood were observed. Conclusion Our findings suggest robust but subtle alterations across different age-groups among ADHD, ASD, and OCD. ADHD-specific ICV and hippocampal alterations in children and adolescents, and ASD-specific cortical thickness alterations in the frontal cortex in adults support previous work emphasizing neurodevelopmental alterations in these disorders

Structural changes during ion channel gating

Ion channels are generally multi-subunit complexes, with the ion conduction pathway formed at the subunit interface. In moving between the closed and open states, three structurally distinct channels, represented by the recently determined structures of a mechanosensitive, ligand-gated and K(+) selective channel, all move transmembrane helices away from the central ion conduction pathway. In all three cases, this results in the displacement of a hydrophobic gate from the ion conduction pathway, freeing ion movement. The channels achieve this by moving the transmembrane helices as rigid bodies using three major types of motion: MscL tilts its helices, the nicotinic ACh receptor rotates its helices, and KirBac1.1 bends its helices. In all cases, the gating motions are likely to take place rapidly. These large and fast movements provide a possible explanation for why the conduction pathways of a wide range of different ion channels are formed at the interface between subunits

Structural themes in ion channels

The recent crystal structure of the prokaryotic inwardly rectifying potassium channel, KirBac1.1, revealed for the first time the structure of a K+ channel in the closed state plus the location of the activation gate. Comparison of the KirBac1.1 structure with other known ion channels reveals a number of common structural features. These common characteristics include the formation of the ion conduction pathway at the interface between adjacent subunits, non-fixed charges forming part of the ion pathway, electrostatic sinks drawing ions into the channel, helix dipoles, and hydrophobic gates that ultimately prevent ion movement. This review describes in detail common structural themes present in ion channels

Modeling of an Ion Channel in Its Open Conformation

We have modeled the structure of KirBac1.1 in an open state using as a starting point the structure of KirBac1.1 in its closed conformation (Protein Data Bank 1P7B). To test the validity of the open-state model, molecular dynamics simulations in octane, a lipid bilayer mimetic, were carried out. Simulations of the closed conformer were used for comparison purposes. The total simulation time was ∼138 ns. The initial open model was refined by using projection maps obtained from electron microscopy experiments on two-dimensional crystals of the inwardly rectifying K+ channel KirBac3.1 from Magentospirillum magnetotacticum captured in its open state (C. Vénien-Bryan, unpublished data). Significant movements of the outer helices take place in going from the closed to the open model in agreement with structural and biochemical data in potassium channels, which suggests that gating is accomplished by a conformational change that takes place in the transmembrane domain upon an external stimulus. The motion of the inner helices is mainly achieved by bending at conserved glycine residues that have been previously reported to act as molecular hinges. Overall, these simulations suggest that the open conformer is stable, providing a plausible all-atom model that will enable the study of potential gating mechanisms in more detail

The N-acyltransferase Lnt: Structure-function insights from recent simultaneous studies

Bacterial lipoproteins have been researched for decades due to their roles in a large number of biological functions. There were no structures of their main three membrane processing enzymes, until 2016 for Lgt and LspA, and then 2017 for Lnt with not one but three simultaneous, independent publications. We have analyzed the recent findings for this apolipoprotein N-acyltransferase Lnt, with comparisons between the novel structures, and with soluble nitrilases, to determine the significance of unique features in terms of substrate's recognition and binding mechanism influenced by exclusive residues, two transmembrane helices, and a flexible loop