Functional Analysis of the Heme and Hemoglobin Binding Domains of SHR (Streptococcal Hemoprotein Receptor)
Date of Award
Master of Science (MS)
Zehava Eichenbaum - Chair
Streptococcus pyogenes (Group A Streptococcus) is a Gram-positive bacterial pathogen that causes significant superficial and invasive diseases. Iron acquisition is an important component of GAS pathogenesis in the human host. The 10 gene sia operon of GAS is involved in the acquisition of iron via heme or heme-binding proteins and encodes an ABC transporter as well as the large multifunctional receptor Shr. Domain analysis of Shr shows that it contains two copies of the DUF1533 (domain of unknown function) in its N-terminal part and two NEAT (NEAr Transporter) domains. NEAT domains are found in variable copy number in surface proteins of Gram-positive pathogens and are implicated in binding to various ligands. A new recombinant Shr protein was cloned and a purification protocol was developed, improving the yield of the full-length protein. A solid phase binding assay was developed and used to demonstrate Shr binding to hemoglobin. Several truncated Shr proteins were expressed and purified: the N-terminal Domain (NTD) up to but not including the first NEAT domain of Shr, the NTD plus the first NEAT domain (NTD-NEAT1) and the second NEAT domain alone (NEAT2). It was determined that Shr’s NTD mediates hemoglobin binding, demonstrating that a new protein pattern in Shr is involved in hemoglobin binding, and implicating the DUF1533 in this process. It was also determined that NTD-N1 and NEAT2 bind heme while NTD does not. Therefore, both NEAT domains may participate in the capture of heme from the host hemoglobin by Shr.
Bentley, Elizabeth Electa, "Functional Analysis of the Heme and Hemoglobin Binding Domains of SHR (Streptococcal Hemoprotein Receptor)." Thesis, Georgia State University, 2009.