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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	Fragment-linking peptide design yields a high-affinity ligand for microtubule-based transport Jessica A. Cross , Magda S. Chegkazi , Roberto Steiner , Derek N. Woolfson , Mark P. Dodding Cell Chemical Biology 26 DOI: 10.1016/j.chembiol.2021.03.010 Show Abstract ▼ Abstract: 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.	Apr 2021
I04-Macromolecular Crystallography	Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors Stefano Pernigo , Magda S. Chegkazi , Yan Y. Yip , Conor Treacy , Giulia Glorani , Kjetil Hansen , Argyris Politis , Soi Bui , Mark P. Dodding , Roberto A. Steiner Elife 7 DOI: 10.7554/eLife.38362 Open Access Show Abstract ▼ Abstract: 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 (KLCTPR). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call ‘Y-acidic’ (tyrosine flanked by acidic residues), in a KLC-isoform specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1TPR 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 KLCTPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity.	Oct 2018
I24-Microfocus Macromolecular Crystallography	The light chains of kinesin-1 are autoinhibited Yan Y. Yip , Stefano Pernigo , Anneri Sanger , Mengjia Xu , Maddy Parsons , Roberto Steiner , Mark P. Dodding Proceedings Of The National Academy Of Sciences 113 (9) DOI: 10.1073/pnas.1520817113 PMID: 26884162 Show Abstract ▼ Abstract: 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.	Feb 2016
I24-Microfocus Macromolecular Crystallography	Structural basis for kinesin-1:cargo recognition Stefano Pernigo , Anneri Lamprecht , Roberto A. Steiner , Mark P. Dodding Science 340 (6130), 356 - 359 DOI: 10.1126/science.1234264 PMID: 23519214 Show Abstract ▼ Abstract: Kinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Structural information on how kinesins recognize their cargoes is required for a molecular understanding of this fundamental and ubiquitous process. Here, we present the crystal structure of the tetratricopeptide repeat domain of kinesin light chain 2 in complex with a cargo peptide harboring a “tryptophan-acidic” motif derived from SKIP (SifA-kinesin interacting protein), a critical host determinant in Salmonella pathogenesis and a regulator of lysosomal positioning. Structural data together with biophysical, biochemical, and cellular assays allow us to propose a framework for intracellular transport based on the binding by kinesin-1 of W-acidic cargo motifs through a combination of electrostatic interactions and sequence-specific elements, providing direct molecular evidence of the mechanisms for kinesin-1:cargo recognition.	Apr 2013

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