Model building, refinement and validation

An introduction to the proceedings of the CCP4 Study Weekend held at the University of Warwick on the 6–7 January 2011

Molecular mechanism for kinesin-1 direct membrane recognition

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\u2013dependent 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\u2013mediated organelle transport

Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients

SKIP controls lysosome positioning using a composite kinesin-1 heavy and light chain-binding domain

The molecular interplay between cargo recognition and regulation of the activity of the kinesin-1 microtubule motor is not well understood. Using the lysosome adaptor SKIP (also known as PLEKHM2) as model cargo, we show that the kinesin heavy chains (KHCs), in addition to the kinesin light chains (KLCs), can recognize tryptophanacidic- binding determinants on the cargo when presented in the context of an extended KHC-interacting domain. Mutational separation of KHC and KLC binding shows that both interactions are important for SKIP-kinesin-1 interaction in vitro and that KHC binding is important for lysosome transport in vivo. However, in the absence of KLCs, SKIP can only bind to KHC when autoinhibition is relieved, suggesting that the KLCs gate access to the KHCs. We propose a model whereby tryptophan-acidic cargo is first recognized by KLCs, resulting in destabilization of KHC autoinhibition. This primary event then makes accessible a second SKIP-binding site on the KHC C-terminal tail that is adjacent to the autoinhibitory IAK region. Thus, cargo recognition and concurrent activation of kinesin-1 proceed in hierarchical stepwise fashion driven by a dynamic network of inter- and intra-molecular interactions

A panchromatic spatially resolved study of the inner 500pc of NGC1052 -- II: Gas excitation and kinematics

We map the optical and near-infrared (NIR) emission-line flux distributions and kinematics of the inner 320$\times$535pc$^2$ of the elliptical galaxy NGC1052. The integral field spectra were obtained with the Gemini Telescope using the GMOS-IFU and NIFS instruments, with angular resolutions of 0''88 and 0''1 in the optical and NIR, respectively. We detect five kinematic components: (1 and 2) Two spatially unresolved components, being a broad line region visible in H$\alpha$, with a FWHM of $\sim$3200km s$^{-1}$ and an intermediate-broad component seen in the [OIII]$\lambda \lambda$4959,5007 doublet; (3) an extended intermediate-width component with 280<FWHM<450km s$^{-1}$ and centroid velocities up to 400km s$^{-1}$, which dominates the flux in our data, attributed either to a bipolar outflow related to the jets, rotation in an eccentric disc or a combination of a disc and large-scale gas bubbles; (4 and 5) two narrow (FWHM<150km s$^{-1}$) components, one visible in [OIII], and one visible in the other emission lines, extending beyond the field-of-view of our data, which is attributed to large-scale shocks. Our results suggest that the ionization within the observed field of view cannot be explained by a single mechanism, with photoionization being the dominant mechanism in the nucleus with a combination of shocks and photoionization responsible for the extended ionization.Comment: Accepted at MNRAS. 17 pages, 17 figure

The light chains of kinesin-1 are autoinhibited

The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLCTPR), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme

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

A small-molecule activator of kinesin-1 drives remodeling of the microtubule network

The microtubule motor kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics