Identification of Conformation Specific Binder for the NA+/Galactose Transporter

Abstract

Na+/galactose transporters (SGLTs) are integral membrane proteins, which co-transport Na+ with sugars from the periplasmic space into the cytoplasm. According to the alternating access model for secondary active transporters, these proteins alternate between outward and inward-facing conformations during the transport cycle. The currently available structures from a bacterial homolog of SGLT from Vibrio parahaemolyticus, (vSGLT) are in the substrate-bound inward-occluded and the substrate-free inward-open conformations. Despite much effort, structures of the outward conformations remain elusive.Isolation of distinct conformations of transporter is a major obstacle for X-ray crystallography due to their conformational heterogeneity. Crystallization chaperones based on various protein scaffolds have emerged as a promising tool to increase the crystallization probability of a selected target protein. Sso7d is a highly stable binding protein derived from the hyperthermophilic archaeon Sulfolobus solfataricus. It has a versatile scaffold for generating binding protein for a wide spectrum of targets. Sso7d-derived proteins are far easier to produce in bacteria and due to their small size, they are capable of targeting areas that are not accessible to standards antibodies.To find binders for the SGLT transporter, we screened an Sso7d-based yeast display library using flow cytometry. After several screening steps, we isolated and purified clones that bind the SGLT protein. We are currently attempting to crystallize a complex of the binder with SGLT. Here we present the characterization of Sso7d-binder from the yeast display library using a two-step procedure involving magnetic and FACS-based screening. The micro-molar interaction between Sso7d-binder and vSGLT was determined by analytical gel filtration, SDS-PAGE, microscale thermophoresis and isothermal titration calorimetry. We anticipate this binder will assist in crystallization of the vSGLT protein. Future screening will utilize transport-impaired mutants to select for an outward-facing conformation, which will provide mechanistic insights of the transport mechanism.