title: Crystal Structure of an Engineered LRRTM2 Synaptic Adhesion Molecule and a Model for Neurexin Binding.
abstract: Synaptic adhesion molecules are key components in development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of chemical synapses. Several families of neuronal adhesion molecules have been identified such as the neuronal cell adhesion molecules, neurexins and neuroligins, and in particular recently several leucine-rich repeat proteins, e.g., Netrin G-ligands, SLITRKs, and LRRTMs. The LRRTMs form a family of four proteins. They have been implicated in excitatory glutamatergic synapse function and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. In addition, LRRTM3 and LRRTM4 have been found to be ligands for heparan sulfate proteoglycans, including glypican. We report here the crystal structure of a thermostabilized mouse LRRTM2, with a Tm 30 °C higher than that of the wild-type protein. We localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation, and prior information about the interaction of the ligand with neurexins, which allowed us to propose a tentative model for the LRRTM-neurexin interaction complex. We also determined affinities of the thermostabilized LRRTM2 and wild-type LRRTM1 and LRRTM2 for neurexin-β1 with and without Ca(2+). Cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapselike contacts. The structural and functional data presented here provide the first structure of an LRRTM protein and allow us to propose a model for the molecular mechanism of LRRTM function in the synaptic adhesion.
authors.affiliation: Institute of Biotechnology, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Anja
authors.initials: A
authors.lastname: Paatero
authors.affiliation: Institute of Biotechnology, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Katja
authors.initials: K
authors.lastname: Rosti
authors.affiliation: Structural Biology Research Centre, VIB , Brussels, Belgium.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Shkumatov
authors.affiliation: Institute of Biotechnology, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Celeste
authors.initials: C
authors.lastname: Sele
authors.affiliation: Neuroscience Center, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Brunello
authors.affiliation: Neuroscience Center, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Kai
authors.initials: K
authors.lastname: Kysenius
authors.affiliation: Institute of Biotechnology, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Prosanta
authors.initials: P
authors.lastname: Singha
authors.affiliation: Department of Materials Science and Engineering, Aalto University , 02150 Espoo, Finland.
authors.forename: Ville
authors.initials: V
authors.lastname: Jokinen
authors.affiliation: Neuroscience Center, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Henri
authors.initials: H
authors.lastname: Huttunen
authors.affiliation: Institute of Biotechnology, University of Helsinki , 00100 Helsinki, Finland.
authors.forename: Tommi
authors.initials: T
authors.lastname: Kajander
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D004330:Drosophila; D057809:HEK293 Cells; D006801:Humans; D007313:Insecta; D008565:Membrane Proteins; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013569:Synapses / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D015816:Cell Adhesion Molecules, Neuronal; C561491:LRRTM2 protein, mouse; D008565:Membrane Proteins; C539628:NRXN1 protein, human; D009419:Nerve Tissue Proteins; D019006:Neural Cell Adhesion Molecules