Cellular mechanotransduction is a key informational system, yet its mechanisms remain elusive. Here we unveil the role of microtubules in mechanosignalling, operating downstream of subnuclear F-actin and nuclear envelope mechanics. Upon mechanical activation, microtubules reorganize from a perinuclear cage into a radial array nucleated by centrosomes. This structural rearrangement triggers degradation of AMOT proteins, which we identify as key mechanical rheostats that sequester YAP/TAZ in the cytoplasm. AMOT is stable in mechano-OFF but degraded in mechano-ON cell states, where microtubules allow AMOT rapid transport to the pericentrosomal proteasome in complex with dynein/dynactin. This process ensures swift control of YAP/TAZ function in response to changes in cell mechanics, with experimental loss of AMOT proteins rendering cells insensitive to mechanical modulations. Ras/RTK oncogenes promote YAP/TAZ-dependent tumorigenesis by corrupting this AMOT-centred mechanical checkpoint. Notably, the Hippo pathway fine-tunes mechanotransduction: LATS kinases phosphorylate AMOT, shielding it from degradation, thereby indirectly restraining YAP/TAZ. Thus, AMOT protein stability serves as a hub linking cytoskeletal reorganization and Hippo signalling to YAP/TAZ mechanosignalling.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Significance Statement Oceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a molecular switch in the FutA protein that allows it to accommodate binding of iron in either the Fe(III) or Fe(II) state using structural biology techniques at room temperature and provide a plausible mechanism for iron binding promiscuity.

Kinesin-1 is a processive dimeric ATP-driven motor that transports vital intracellular cargos along microtubules (MTs). If not engaged in active transport, kinesin-1 limits futile ATP hydrolysis by adopting a compact autoinhibited conformation that involves an interaction between its C-terminal tail and the N-terminal motors domains. Here, using a chimeric kinesin-1 that fuses the N-terminal motor region to the tail and a tail variant unable to interact with the motors, we employed high-resolution cryo-EM in the presence of MTs to investigate elements of the mechanochemical cycle. We describe a missing structure for the proposed two-step allosteric mechanism of ADP release, the ATPase rate limiting step. It shows that MT association induces remodeling of the hydrogen bond network at the nucleotide binding site triggering removal of the Mg2+ ion from the Mg2+-ADP complex resulting in a strong MT-binding apo-like state before ADP dissociation. We further demonstrate that tail association does not directly affect this mechanism, nor the adoption of the ATP hydrolysis-competent conformation, nor neck linker docking/undocking, even when zippering the two motor domains. Based on this structural evidence, we propose a revised mechanism for tail-dependent kinesin-1 autoinhibition and suggest a possible explanation for its characteristic pausing behavior on MTs. ### Competing Interest Statement The authors have declared no competing interest.

Hydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand-recognition processes and protein–protein interactions. However, their direct visualization by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localization of only the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher). However, many H atoms of biochemical significance remain undetectable by this method. In contrast, neutron diffraction methods enable the visualization of most H atoms, typically in the form of deuterium (2H) atoms, at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought. REFMAC5 from the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP4 Monomer Library, the source of prior chemical information used in the refinement. One new feature for neutron data analysis in REFMAC5 is refinement of the protium/deuterium (1H/2H) fraction. This parameter describes the relative 1H/2H contribution to neutron scattering for hydrogen isotopes. The newly developed REFMAC5 algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) using either (i) neutron data only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement with REFMAC5 afforded models characterized by R-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium–low resolution.

Striated muscle sarcomeres operate by sliding of the regular arrays of interdigitating thin (actin) and thick (myosin) filaments. Thick filaments are held together at their centre by M-band bridges, maintaining sarcomere structure during contraction and relaxation. The giant sarcomeric protein titin is instrumental in maintaining order, since C-terminal truncations of titin lead to loss of A-band alignment and sarcomere failure. The C-terminal sequence of titin encodes a kinase domain, 10 Ig domains and linker sequences, and is associated with the bare zone at the centre of the sarcomere. Unlike the cross-bridge associated sequence, it does not show any correlation with the organisation of the thick filament bare zone and associated M-band bridges. The precise disposition of titin remains unclear, e.g. whether anti-parallel molecules overlap at centre of the filament. We used superresolution light microscopy with new titin antibodies to establish the position of several titin epitopes in the bare zone, together with key markers for the M-band proteins myomesin and M-protein. We show that the titin kinase domain is at the edge of the bare zone, about 70 nm from the M-band centre at the first myosin motor domains and may therefore account for the first of the accessory protein stripes in the A-band. SAXS data of two titin constructs covering several domains of the bare-zone associated sequence, one from the beginning including the kinase and the other from the end, suggest that they are extended structures but with compact linkers. Conversely, the long insert sequence between M-band Ig-domains 3 and 4 (Ig-162 and Ig-163) appears to be more extensive. Our combined data suggests that in the bare zone, titin molecules from each side of the thick filament overlap each other by the width of the M-band.

Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the & alpha;/& beta;-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses under normoxic and hyperoxic conditions with molecular dynamics and quantum mechanical calculations to investigate its prototypic 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) member. O2 localization to the "oxyanion hole", where catalysis occurs, is an unfavorable event and the direct competition between dioxygen and water for this site is modulated by the "nucleophilic elbow" residue. A hydrophobic pocket that overlaps with the organic substrate binding site can act as a proximal dioxygen reservoir. Freeze-trap pressurization allowed the structure of the ternary complex with a substrate analogue and O2 bound at the oxyanion hole to be determined. Theoretical calculations reveal that O2 orientation is coupled to the charge of the bound organic ligand. When 1-H-3-hydroxy-4-oxoquinaldine is uncharged, O2 binds with its molecular axis along the ligand's C2-C4 direction in full agreement with the crystal structure. Substrate activation triggered by deprotonation of its 3-OH group by the His-Asp dyad, rotates O2 by approximately 60 & DEG;. This geometry maximizes the charge transfer between the substrate and O2, thus weakening the double bond of the latter. Electron density transfer to the O2(& pi;*) orbital promotes the formation of the peroxide intermediate via intersystem crossing that is rate-determining. Our work provides a detailed picture of how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish metal-independent oxygenation. We report how the common & alpha;/& beta;-hydrolase architecture and its simple catalytic machinery have been adapted to accomplish spin-restricted metal-independent oxygenation.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property which we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H 2 O 2 -oxidative conditions. Cysteine enrichment was concurrent with transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with β-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

AbstractHydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand recognition processes, and protein-protein interactions. However, their direct visualisation by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localisation of the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher), that is not often attainable. Differently, neutron diffraction methods enable the visualisation of most H atoms, typically in the form of deuterium (D) atoms at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought.REFMAC5 of the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of theCCP4 Monomer Library, the source of prior chemical information used in refinement. One new feature for neutron data analysis inREFMAC5 is the refinement of the protium/deuterium (1H/D) fraction. This parameter describes the relative1H/D contribution to neutron scattering for H atoms. The newly developedREFMAC5algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) by using either (i) neutron data-only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement withREFMAC5afforded models characterised byR-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy especially for structures at medium-low resolution.SynopsisThe macromolecular refinement packageREFMAC5 of theCCP4 suite has been extended with the incorporation of algorithms for neutron crystallography.

Room-temperature biological crystallography has seen a resergence in recent years and a collection of articles recently published in IUCrJ, Acta Cryst. D Structural Biology and Acta Cryst. F Structural Biology Communications, have been collected together to produce a virtual special issue at https://journals.iucr.org/special_issues/2022/RT/.

Hydrogen (H) atoms represent a large fraction of the total atomic content of macromolecules.They often play critical roles in enzymatic reactions, as they are involved in the protonation of active site residues, in the organization and orientation of the solvent network, and in the establishment of hydrogen bonds.However, the direct visualisation of H atoms in macromolecules is not straightforward.Since their contribution to the total scattering is minimal owing to their low electron content, only a small percentage of H atoms can be located using X-ray macromolecular crystallography (MX), even at sub-atomic resolution (<1.2 Å).In contrast with MX, neutron macromolecular crystallography (NMX) relies on the interaction between neutrons and atomic nuclei.Ratio of atomic scattering factors of deuterium (D) to that of typical heavy macromolecular atoms such as oxygen, carbon and nitrogen is not far from 1 (1.15, 1.08 and 0.71 respectively).Therefore, the observation of H atoms (typically in the form of D) is possible even at modest resolution (2.0-2.5 Å).Despite the major limitations of this technique, especially due to the low flux of neutron beams, NMX studies are particularly useful in answering research questions that require specific knowledge of H atom positions with the view of obtaining mechanistic insight.Whilst NMX maps show the nuclear positions of H atoms, MX maps indicate the positions of valence-electron density for H atoms shifted along their bond vector.Cryo-electron microscopy (cryo-EM) and electron diffraction experiments inform on both nuclear and electron localisation of H atoms.This project aims at modelling and refining H atoms by using different experimental data (cryo-EM, NMX and electron diffraction) integrated in a common framework, to provide new insights in biological processes such as enzyme mechanisms.This contribution will describe the recent developments in the crystallographic refinement package REFMAC5 [1], for the refinement of structural models obtained by NMX data.The CCP4 Monomer Library [2], typically used as the source for prior chemical information in REFMAC5 and other programs such as Coot [3], has been recently extended to include accurate H atom nucleus to parent atom distances derived from neutron data analysis [4] and quantum mechanics (QM) calculations.New features for the refinement of mixture H/D parameters in NMX and the use of reference structure restraints will be presented.

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

Adhesion of basal keratinocytes to the underlying extracellular matrix (ECM) plays a key role in the control of skin homeostasis and response to injury. Integrin receptors indirectly link the ECM to the cell cytoskeleton through large protein complexes called focal adhesions (FA). FA also function as intracellular biochemical signaling platforms to enable cells to respond to changing extracellular cues. The α4β1 and α9β1 integrins are both expressed in basal keratinocytes, share some common ECM ligands, and have been shown to promote wound healing in vitro and in vivo. However, their roles in maintaining epidermal homeostasis and relative contributions to pathological processes in the skin remain unclear. We found that α4β1 and α9β1 occupied distinct regions in monolayers of a basal keratinocyte cell line (NEB-1). During collective cell migration (CCM), α4 and α9 integrins co-localized along the leading edge. Pharmacological inhibition of α4β1 and α9β1 integrins increased keratinocyte proliferation and induced a dramatic change in cytoskeletal remodeling and FA rearrangement, detrimentally affecting CCM. Further analysis revealed that α4β1/α9β1 integrins suppress extracellular signal-regulated kinase (ERK1/2) activity to control migration through the regulation of downstream kinases including Mitogen and Stress Activated Kinase 1 (MSK1). This work demonstrates the roles of α4β1 and α9β1 in regulating migration in response to damage cues.

Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O 2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O 2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H 2 O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH 3 + side chain engaged in a strong hydrogen bond with Thr57 OG1 , while the Thr57 OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHU C5 with Thr57 HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O 2 exchange during catalysis.

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1-dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1-mediated organelle transport.

Hydrogen (H) atoms often play essential roles in enzymatic reactions as they are responsible for the reversible protonation of active site residues and for the organisation of the solvent network.More generally, H atoms are also necessary for the establishment of Hbonds which, in turn, stabilise interactions between macromolecules and between macromolecules and their ligands.Although H atoms represent a large fraction of the total atomic content of macromolecules their direct visualisation is not straightforward.Even at (sub-)atomic resolution (<1.2 Å), X-ray macromolecular crystallography (MX), the most common technique for structural determination, affords the localisation of only a small percentage of H atoms as their contribution to the total scattering is minimal owing to their low electron content.Differently from MX, neutron macromolecular crystallography (NMX) relies on the interaction between neutrons and atomic nuclei.With this technique the visualisation of H atoms (typically in the form of deuterons, D) is possible even at modest resolution (2.0-2.5 Å), making it an ideal complementary tool to MX when H localisation is deemed important.Whilst NMX maps show the nuclear positions of H atoms, MX maps indicate the positions of valence-electron density for H atoms shifted along their bond vector.Cryo-electron microscopy (cryo-EM) and electron diffraction experiments inform on both nuclear and electron localisation of H atoms.This project aims at modelling and refining H atoms by using different experimental data (cryo-EM, NMX and electron diffraction) integrated in a common framework, to provide new insights in biological processes such as enzyme mechanisms.New features in the crystallographic refinement package REFMAC5 [1], one of the flagships of the scientific CCP4 computational suite, have been developed and will be presented.The CCP4 Monomer Library [2], typically used as the source for prior chemical information in REFMAC5 and other programs such as COOT [3], has been recently implemented for more accurate H atom positions derived from neutron data analysis [4] and quantum mechanics (QM) calculations.Recent developments in REFMAC5 and tools for the refinement of structural models obtained by neutron diffraction data will be presented.

Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides that make improved interactions with the target. Here, we devise a fragment-linking strategy-"mash-up" design-to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolar-affinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.

Dermatofibromas are common benign skin lesions, the etiology of which is poorly understood. We identified two unrelated pedigrees in which there was autosomal dominant transmission of multiple dermatofibromas. Whole exome sequencing revealed a rare shared heterozygous missense variant in the F13A1 gene encoding factor XIII subunit A (FXIII-A), a transglutaminase involved in hemostasis, wound healing, tumor growth, and apoptosis. The variant (p.Lys679Met) has an allele frequency of 0.0002 and is predicted to be a damaging mutation. Recombinant human Lys679Met FXIII-A demonstrated reduced fibrin crosslinking activity in vitro. Of note, the treatment of fibroblasts with media containing Lys679Met FXIII-A led to enhanced adhesion, proliferation, and type I collagen synthesis. Immunostaining revealed co-localization between FXIII-A and α4β1 integrins, more prominently for Lys679Met FXIII-A than the wild type. In addition, both the α4β1 inhibitors and the mutation of the FXIII-A Isoleucine-Leucine-Aspartate-Threonine (ILDT) motif prevented Lys679Met FXIII-A-dependent proliferation and collagen synthesis of fibroblasts. Our data suggest that the Lys679Met mutation may lead to a conformational change in the FXIII-A protein that enhances α4-integrin binding and provides insight into an unexpected role for FXIII-A in the pathobiology of familial dermatofibroma.