Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors

The light chains (KLCs) of the heterotetrameric microtubule motor kinesin-1, that bind to cargo adaptor proteins and regulate its activity, have a capacity to recognize short peptides via their tetratricopeptide repeat domains (KLC TPR ). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call \u2018Y-acidic\u2019 (tyrosine flanked by acidic residues), in a KLC-isoform specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1 TPR are distinct from those utilized for the recognition of W-acidic motifs found in adaptors that are KLC-isoform non-selective. However, a partial overlap on their receptor binding sites implies that adaptors relying on Y-acidic and W-acidic motifs must act independently. We propose a model to explain why these two classes of motifs that bind to the concave surface of KLC TPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity

Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD

New insight into cofactor-free oxygenation from combined experimental and computational approaches:Multi-protein assemblies in signaling • Catalysis and regulation

Molecular oxygen (O2), in spite being a potentially strong oxidant, typically displays very poor reactivity with organic molecules. This is largely due to quantum chemical reasons as O2 in its ground state is a diradical (3O2) whilst common organic substrates are in a singlet state. For this reason catalysis involving O2 as a reactant is typically mediated by enzymes containing redox metal and/or organic co-factors. Cofactor-independent oxygenases (and oxidases) are therefore intriguing enzymes from a fundamental viewpoint. This review looks at recent advances that have been made in understanding of this class of intriguing biocatalysts highlighting the power of an inter-disciplinary approach involving structural biology, spectroscopy and theoretical methods

Online Raman spectroscopy for Structural Biology on beamline ID29 of the ESRF

International audienceRaman spectroscopy can probe the structure and conformations of specific chemical groups within proteins and may thus be used as a technique complementary to X-ray crystallography. This combined approach can be decisive in resolving ambiguities in the interpretation of enzymatic or X-ray induced processes. Here, we present an online Raman setup developed at the European Synchrotron that allows for interleaved Raman spectra acquisition and X-ray diffraction measurements with fast probe exchange and simple alignment while maintaining a high sensitivity over the entire spectral range. This device has been recently employed in the study of a covalent intermediate in the O2-dependent breakdown of uric acid by the cofactor-free enzyme urate oxidase and to monitor its decay induced by X-ray exposure

Evolutionary adaptation from hydrolytic to oxygenolytic catalysis at the α/β-hydrolase fold

Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the α/β-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°. 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(π*) 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

The Mycobacterium tuberculosis cytochromes P450: Physiology, biochemistry & molecular intervention

The human pathogen Mycobacterium tuberculosis (Mtb) encodes 20 cytochrome P450 (P450) enzymes. Gene essentiality for viability or host infection was demonstrated for Mtb P450s CYP128, CYP121 and CYP125. Structure/function studies on Mtb P450s revealed key roles contributing to bacterial virulence and persistence in the host. Various azole-class drugs bind with high affinity to the Mtb P450 heme and are potent Mtb antibiotics. This paper reviews the current understanding of the biochemistry of Mtb P450s, their interactions with azoles and their potential as novel Mtb drug targets. Mtb multidrug resistance is widespread and novel therapeutics are desperately needed. Simultaneous drug targeting of several Mtb P450s crucial to bacterial viability/persistence could offer a new route to effective antibiotics and minimize the development of drug resistance. </jats:p

Origin of the Proton-transfer Step in the Cofactor-free (1H)-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

Dioxygenases catalyze a diverse range of chemical reactions that involve the incorporation of oxygen into a substrate and typically use a transition metal or organic cofactor for reaction. Bacterial 1-H-3- hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) belongs to a class of oxygenases able to catalyze this energetically unfavourable reaction without any cofactor. In the quinaldine metabolic pathway HOD breaks down its natural N-heteroaromatic substrate using a mechanism that is still incompletely understood. Experimental and computational approaches were combined to study the initial step of the catalytic cycle. We have investigated the role of the active site His251/Asp126 dyad, proposed to be involved in substrate hydroxyl group deprotonation, a critical requirement for subsequent oxygen reaction. The pH profiles obtained under steady-state conditions for the H251A and D126A variants show a strong pH effect on their kcat and kcat/Km constants, with a decrease in kcat/Km of 5500-fold and 9-fold at pH 10.5, respectively. Substrate deprotonation studies under transient-state conditions show that this step is not rate limiting and yield a pKa value of ~7.2 for wt HOD. A large solvent isotope effect was found and the pKa value was shifted to ~8.3 in D2O. Crystallographic and computational studies reveal that the mutations have minor effect on substrate positioning. Computational work shows that both His251 and Asp126 are essential for the proton transfer driving force of the initial reaction. This multi-disciplinary study offers unambiguous support to the view that substrate deprotonation, driven by the His/Asp dyad, is an essential requirement for its activation

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

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