CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

Electric fan use for cooling during hot weather: a biophysical modelling study.

BACKGROUND: In hot weather, electric fans can potentially provide effective cooling for people, with lower greenhouse gas emissions and cost than air conditioning. However, international public health organisations regularly discourage fan use in temperatures higher than 35°C, despite little evidence. We aimed to determine humidity-dependent temperature thresholds at which electric fans would become detrimental in different age groups. METHODS: We used biophysical modelling to determine the upper humidity-dependent temperature thresholds at which fan use would become detrimental (ie, worsen heat stress) for healthy young adults (aged 18-40 years), healthy older adults (aged ≥65 years), and older adults taking anticholinergic medication. We also obtained hourly environmental data for the period Jan 1, 2007, to Dec 31, 2019, for 108 populous cities to determine the number of days fan use would be effective for cooling, standardised to a 31-day hot weather month. We established simplified temperature thresholds for future fan use recommendations on the basis of temperatures below which fan use would never have been detrimental between Jan 1, 2007, and Dec 31, 2019, across all prevailing levels of ambient humidity. FINDINGS: According to our model, fan use would have been beneficial on 30·0 (96·6%) of 31 hot weather days for healthy young adults and 29·4 (94·9%) of 31 hot weather days for both older adults and older adults taking anticholinergic medication between Jan 1, 2007, and Dec 31, 2019. Adherence to the current WHO recommendation of fan use below temperatures of 35°C only, fan use would have been recommended on 27·2 days (87·7%) of 31 hot weather days. According to our simplified thresholds for fan use (at temperatures <39·0°C for healthy young adults, <38·0°C for healthy older adults, and <37·0°C for older adults taking anticholinergic medication), fan use would have been recommended on 29·6 (95·5%) of 31 hot weather days in healthy young adults, 29·4 (94·8%) days in healthy older adults, and 28·8 (93·0%) days in older adults taking anticholinergic medication between Jan 1, 2007, and Dec 31, 2019. INTERPRETATION: Electric fan use, particularly for healthy young adults, would not have worsened heat stress on the majority of study days between 2007 and 2019. Our newly proposed thresholds for fan use provide simple guidelines that improve future heatwave fan use recommendations. FUNDING: None.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.

Hot new structures of the cold sensor, TRPM8, reveal insights into the fundamentals of cold perception and adaptation.

The ion channel TRPM8 has been identified as the primary cold sensor in humans, but insights into how cold perception is established remained elusive. This has changed now with newly published structures of TRPM8 in different states during channel gating, which help us understand why we feel cold and adapt to it.

Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.

KEY POINTS: Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na+ , K+ and Ca2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K+ channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. ABSTRACT: Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K+ channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway.

An allosteric binding site of the α7 nicotinic acetylcholine receptor revealed in a humanized acetylcholine-binding protein.

Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine-binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the ß8-ß9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel-mediated disorders.

Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane ß-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

Conserved Omp85 lid-lock structure and substrate recognition in FhaC.

Omp85 proteins mediate translocation of polypeptide substrates across and into cellular membranes. They share a common architecture comprising substrate-interacting POTRA domains, a C-terminal 16-stranded ß-barrel pore and two signature motifs located on the inner barrel wall and at the tip of the extended L6 loop. The observation of two distinct conformations of the L6 loop in the available Omp85 structures previously suggested a functional role of conformational changes in L6 in the Omp85 mechanism. Here we present a 2.5 Å resolution structure of a variant of the Omp85 secretion protein FhaC, in which the two signature motifs interact tightly and form the conserved 'lid lock'. Reanalysis of previous structural data shows that L6 adopts the same, conserved resting state position in all available Omp85 structures. The FhaC variant structure further reveals a competitive mechanism for the regulation of substrate binding mediated by the linker to the N-terminal plug helix H1.

The structural basis of autotransporter translocation by TamA.

TamA is an Escherichia coli Omp85 protein involved in autotransporter biogenesis. It comprises a 16-stranded transmembrane ß-barrel and three POTRA domains. The 2.3-Å crystal structure reveals that the TamA barrel is closed at the extracellular face by a conserved lid loop. The C-terminal ß-strand of the barrel forms an unusual inward kink, which weakens the lateral barrel wall and creates a gate for substrate access to the lipid bilayer.