The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin

Abstract

Staphylococcus aureus is a medically important bacterial pathogen that during infections acquires iron from human hemoglobin (Hb). It uses two closely related iron regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAr iron Transporter (NEAT) domains connected by a helical linker domain. To gain insight into the mechanism of extraction we used NMR to investigate the structure and dynamics of the 38.8 kDa tri-domain IsdH protein (IsdHN2N3, A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small angle x-ray scattering, residual dipolar coupling and inter-domain NOE data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface, and based on NMR relaxation data undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and -bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced fit mechanisms that may promote hemin release from Hb by altering the position of its F-helix.

An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins

“…Paramagnetic metals can occur naturally in a macromolecule (Butler 1998; Sjodt et al 2015) or be introduced by replacing an existing metal (Keniry et al 2006), covalently or non-covalently added as a chelate (Huang et al 2013; Lee et al 2015; Loh et al 2015; Wei et al 2013; Yang et al 2015), bound by an unnatural amino acid (Loh et al 2013; Park et al 2015), or lanthanide-binding peptide sequence tag (LBT) engineered into the protein sequence (Barthelmes et al 2011; Feeney et al 2001; Gaponenko et al 2000; Martin and Imperiali 2015; Nitz et al 2003; Su et al 2008), to reference a few key examples. One common limitation to artificial tags is the presence of metal ion motion.…”

Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies

“…B. Representative [ 1 H- 15 N]-HSQC spectrum of IsdH N2N3 (see Sjodt et al, 2016 for more information). Boxed regions indicate the representative sections of each PRE probe's spectra shown in (C) (K499R1 and E559R1).…”

“…A representative calibration curve used for the conversion of intensity ratios into distances for the NMR study of IsdH N2N3 (Sjodt et al, 2016). The curve was generated according to Equations 1 and 2 using an average linewidth (π/R2) and correlation time (τc) of 23 Hz and 16.4 ns, respectively.…”

“…Site-specific attachment of paramagnetic spin labels to proteins enhances the transverse relaxation rates of nearby nuclei leading to line-broadening effects that can be used to derive distance restraints (Battiste and Wagner, 2000; Iwahara et al , 2004; Clore and Iwahara, 2009). PRE-derived distance restraints have been used to characterize the structures of various molecules, including amongst others membrane proteins (Roosild et al , 2005), multi-domain proteins displaying inter-domain dynamics (Sjodt et al , 2016), single domain proteins (Battiste and Wagner, 2000), protein-DNA complexes (Clore and Iwahara, 2009), transient protein-protein interactions (Tang et al , 2007; Villareal et al , 2011), and intrinsically disordered proteins (Bertoncini et al , 2005). Two approaches are generally used to obtain PRE-derived distance restraints using proteins that are labeled with paramagnetic probes.…”

“…Note: These reagents were used to obtain the PRE data according to Sjodt et al, 2016. However, other brands of these materials may be used if necessary.…”