Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.

Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter.

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na+ and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an 'elevator-with-an-operator' mechanism.

Selective Nutrient Transport in Bacteria: Multicomponent Transporter Systems Reign Supreme.

Multicomponent transporters are used by bacteria to transport a wide range of nutrients. These systems use a substrate-binding protein to bind the nutrient with high affinity and then deliver it to a membrane-bound transporter for uptake. Nutrient uptake pathways are linked to the colonisation potential and pathogenicity of bacteria in humans and may be candidates for antimicrobial targeting. Here we review current research into bacterial multicomponent transport systems, with an emphasis on the interaction at the membrane, as well as new perspectives on the role of lipids and higher oligomers in these complex systems.

Perceptual harmony in judgments of group prototypicality and intragroup respect.

We test common sense psychology of intragroup relations whereby people assume that intragroup respect and ingroup prototypicality are positively related. In Study 1a, participants rated a group member as more prototypical if they learned that group member was highly respected rather than disrespected. In Study 1b, participants rated a group member as more respected by other group members if they learned that group member was prototypical rather than unprototypical. As a commonsense psychology of groups, we reasoned that the perceived relationship between prototypicality and intragroup respect would be stronger for cohesive groups compared to incohesive groups. The effect of intragroup respect on perceptions of prototypicality (Study 2a & 2c) and the effect of prototypicality on perceptions of intragroup respect (Study 2b) were generally stronger for participants considering cohesive groups relative to incohesive groups. However, the interaction effect of prototypicality and group cohesion on intragroup respect did fail to replicate in Study 2d. In Studies 3, 4a, and 4b we manipulated the relationship between prototypicality and intragroup respect and found that when these variables were in perceptual harmony participants perceived groups as more cohesive. The results of eight out of nine studies conducted are consistent with the prediction that people make inferences about intragroup respect, prototypicality, and group cohesion in a manner that maintains perceptual harmony.

Identifying the "Therapy Targets" for Treating the Negative Symptoms of Psychosis Using Cognitive Behavioral Therapy.

The division of psychotic symptoms into positive and negative categories has largely divided the research on them. While the research on positive symptoms of psychosis has rapidly developed over the last three decades, the literature on negative symptoms has noticeably lagged behind. Negative symptoms have likely been ignored in the treatment literature because they were previously thought to remit following the treatment of positive symptoms. Recent evidence does not consistently support this theory and indicates that the different manifestations of negative symptoms require distinct approaches to treatment. The current review provides a re-evaluation of the theoretical literature on negative symptoms to inform and identify "treatment targets" to reduce them. The "treatment targets" are then translated into intervention strategies using a cognitive behavioral framework. A review of the empirical literature on cognitive behavior therapy for treating negative symptoms is then offered along with a critical discussion of where cognitive behavior therapy stands compared to other interventions and what research is still needed.

Sleep and Mental Health in the General Population of Elderly Women.

Sleep and mental health complaints are prevalent in the elderly and share common risk factors. We assessed the relationship between sleep and mental health in three representative samples of elderly women while controlling for multiple risk factors common to both. We performed this cross sectional secondary data analysis in 2015 using 2013 data from the Behavioral Risk Factor Surveillance System (BRFSS) for females ages 65 years and older from California (N = 1912), Florida (N = 9120), and Pennsylvania (N = 2429). We conducted multiple logistic regression analysis to assess the relationship between sleep duration group (short, moderate/reference, or long) and mental health issues in the past 30 days (yes or no) in elderly females, while controlling for multiple covariates. About 25% of the elderly females reported mental health issues and 20% reported short or long sleep durations. In adjusted analysis, compared to the elderly females in the moderate sleep duration group (averaging 6-8 h of sleep per day), those in the short and long sleep duration groups had increased prevalence of mental health issues by 66% and 26%, respectively. Mental health was also related to physical health issues including general health status, activity limitations, and chronic health conditions. Overall, sleep was related to mental health in representative samples of elderly females even after controlling for risk factors common to both. Even though we could not determine the direction of influence, the findings indicate a need for clinicians to screen their elderly female patients for both sleep and mental health issues, especially in those with physical health comorbidities.

Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator. The ubiquitin E3 ligases provide specificity to the cascade and often bind the substrate, while the ubiquitin-conjugating enzymes (E2s) have a pivotal role in determining chain linkage and length. Interaction of ubiquitin with the E2 is important for activity, but the weak nature of these contacts has made them hard to identify and study. By reviewing available crystal structures, we identify putative ubiquitin binding sites on E2s, which may enhance E2 processivity and the assembly of chains of a defined linkage. The implications of these new sites are discussed in the context of known E2-ubiquitin interactions.

Noncovalent Ubiquitin Interactions Regulate the Catalytic Activity of Ubiquitin Writers.

Covalent modification of substrate proteins with ubiquitin is the end result of an intricate network of protein-protein interactions. The inherent ability of the E1, E2, and E3 proteins of the ubiquitylation cascade (the ubiquitin writers) to interact with ubiquitin facilitates this process. Importantly, contact between ubiquitin and the E2/E3 writers is required for catalysis and the assembly of chains of a given linkage. However, ubiquitin is also an activator of ubiquitin-writing enzymes, with many recent studies highlighting the ability of ubiquitin to regulate activity and substrate modification. Here, we review the interactions between ubiquitin-writing enzymes and regulatory ubiquitin molecules that promote activity, and highlight the potential of these interactions to promote processive ubiquitin transfer.

Secondary ubiquitin-RING docking enhances Arkadia and Ark2C E3 ligase activity.

RING-domain E3 ligases enhance transfer of ubiquitin to substrate proteins by stabilizing the RING-bound thioester-linked E2∼ubiquitin conjugate in a defined conformation that primes the active site for nucleophilic attack. Here we report that the monomeric RING domains from the human E3 ligases Arkadia and Ark2C bind directly to free ubiquitin with an affinity comparable to that of other dedicated ubiquitin-binding domains. Further work showed that the Ark-like RING domain and the noncovalently bound ubiquitin molecule coordinately stabilize the E2-conjugated ubiquitin (donor ubiquitin) in the 'closed' conformation. Our studies identify the RING domain of Arkadia as a ubiquitin-binding domain and provide insight into a new ubiquitin-dependent mechanism used by monomeric RING domains to activate ubiquitin transfer. This study also suggests how substrates that have been monoubiquitinated could be favored for further ubiquitination.