Structural and biophysical studies of the mammalian Na+ dependent Cl--HCO3-exchanger NCBE and the bacterial enzyme isatin hydrolase

Abstract

Part I. Acid-base homeostasis is fundamental to our understanding of human physiology and is essential to cellular function. The main buffering system found in the human body is based on bicarbonate. The SLC4 proteins are the main facilitators of bicarbonate transport across the plasma membrane, however, not much is known about the structural basis of function and regulation of these. In the work described in this thesis, the N-terminal cytoplasmic domain (NTD) of the sodium-coupled chloride bicarbonate exchanger (NCBE), found predominantly in the choroid plexus of the brain, has been cloned, expressed and purified. The core domain found centrally in the NTD has been crystallized and the structure determined to 4.0 Å resolution. The NTD of NCBE is found to contain regions of intrinsic protein disorder and these disordered regions are conserved among all bicarbonate transporters of the SLC4 family. The disordered regions coincide with regions of sequence variation, indicating that although sequence is not conserved, the disorder is. A protocol for identification of small molecule ligands for disordered regions is described. The NTD is found to contain several zinc binding sites and one magnesium binding site. The effect of zinc binding along with the NTDs response to pH variation is assessed by circular dichroism and chromatographic techniques. Part II. Isatin hydrolase is a part of a indole-3-acetic acid degradation pathway in bacteria. The structural basis of the enzyme is unknown and the fold has not previously been described although structural homologues are known. In this thesis, the crystal structures of apo isatin hydrolase and a complex with product analogue thioisatinate are described. The structural analysis enables stronger identification of structural homologues such as bacterial kynurenine formamidase. Structural homologues of low sequence identity are also identified in eukaryotes and in multi-domain proteins. The crystals structures of isatin hydrolase also reveal a transient water wire that may facilitate proton transfer during hydrolysis of isatin. The function of a key serine coordinating the water wire is tested through mutagenesis. Isatin hydrolase is characterized biophysically in order to understand the temperature, pH and co-factor requirements for the successful application in a biotechnological role. A protocol for application of isatin hydrolase in a simple and highly selective tool for quantification of isatin in blood samples is described.