CommunicationNMR experiments on the transient interaction of the intrinsically disordered N-terminal peptide of cystathionine-β-synthase with heme
Graphical abstract
Introduction
Intrinsically disordered proteins (IDP) and protein regions (IDPR) attached to folded protein domains have been found to be implicated in critical biological functions and in neurodegenerative and other diseases [1], [2], [3], [4], [5]. In contrast to covalent binding, as in many metalloproteins, IDPs have been shown to be involved in functionally important transient interactions. In many cases, heme-binding (HBM) or heme-regulatory motifs (HRM) with special amino acid combinations such as CxxHx18H, CxxHx16H and cysteine-proline (CP) in small sequence stretches are responsible for the heme association with moderate binding constants [6], [7], [8], [9], [10]. Coupled with mutagenesis, the heme-protein interaction studies are often carried out via techniques such as resonance Raman, surface plasmon resonance, NMR, circular dichroism (CD) and UV–Vis spectroscopy [11], [12], [13]. Such studies demonstrated the possibility for the IDPR to undergo conformational changes upon heme binding, e.g. due to hexa-coordination of the iron moiety in the iron-porphyrin complex with cysteine and histidine residues located at different positions in the protein sequence, leading to a reduction in the flexibility of the polypeptide chain [8], [9], [10]. A variety of techniques like chemical shift perturbation, relaxation dispersion spectroscopy and paramagnetic relaxation enhancement have been demonstrated for studies of protein-ligand interactions [14], [15], [16], [17], [18] and [1H, 15N]-HSQC experiments are often employed to map the interaction interface. However, in experimental situations leading to fast amide proton exchange with water molecules, e.g. at physiological temperature and pH, its applicability may be limited [19]. Thus, the required protocol strongly depends on the given experimental situation and the use of 13C direct detection experiments [20], [21], [22], [23] has been shown to offer an efficient alternative approach for structural investigations of uniformly enriched proteins and their complexes.
Many IDPs are involved in regulatory interactions with heme, with Cys-Pro (CP) motifs often playing an important role [8], [9], [10]. The proline residue in the CP motif assists the coordination of cysteine thiolate to the Fe(III) heme complex [12], [24]. We are currently investigating the N-terminal peptide stretch (1–40) of human cystathionine-β-synthase (CBS, 551 a.a.; UniProtKB P35520) in the context of heme-IDP interaction [25], [26], [27]. In the enzyme CBS heme is bound as a cofactor via an axial coordination to cysteine-52 and histidine-65. However, NMR and UV/Vis studies [28] have shown that the disordered N-terminal region of CBS also contributes heme-binding capacities via a second binding site, the CP-based motif around cysteine-15 possibly extending to histidine-22. Functional assays revealed that this second heme-binding site increases the efficacy of the enzyme by ∼30%.
Taking into consideration these factors, we aimed at a further robust strategy for probing the transient interaction of the N-terminal peptide stretch of human cystathionine-β-synthase (CBS(1–40)) with heme.
Section snippets
Materials and methods
CBS(1–40) (0.6 mM) was expressed via a fusion protein approach with the streptococcal protein GB1. The sample preparation followed protocols described earlier [29]. Heme was dissolved 1 mM in 20 mM NaOH and incubated for 30 min. NMR measurements on CBS(1–40) were performed in sodium phosphate buffer pH 6.9 and carried out on a Bruker 600 MHz narrow-bore Avance III NMR spectrometer equipped with pulse field gradient accessories, pulse shaping units and triple resonance cryo-probe with the sample
Results
Initial heme-binding studies were carried out via [1H, 15N]-HSQC experiments. To improve the solubility of the target protein, make use of easy purification protocols, and minimize proteolytic degradations, GB1 fusion peptide samples of the N-terminal (1–40) of CBS (wild type and mutants) were employed in these investigations, and it is seen that the N-terminal peptide upon heme binding undergoes transient formation of a hexa-coordinated complex that is sparsely populated and in exchange with
Discussion and conclusions
The objective of this study was to evaluate the heme-binding of the non-crystallizable N-terminal peptide stretch (1–40) of human cystathionine-β-synthase, an intrinsically disordered protein region. As the conventional approach via [1H, 15N]-HSQC interaction mapping experiments was hampered by fast HN exchange, the HCBCACON protocol was adapted as an alternative strategy. This experiment has the combined advantages of being HN exchange-independent and of adding proline correlations as
Compliance with ethical standards
Funding: This work was supported by the Deutsche Forschungsgemeinschaft (DFG) [grant number: FOR 1738 (OH 86/3-2)].
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgments
We thank S. Häfner (CS Protein production, FLI) for technical support. Financial support by the Deutsche Forschungsgemeinschaft (DFG) within FOR 1738 (to D.I. and O.O.) is gratefully acknowledged. The FLI is a member of the Leibniz Association (WGL) and is financially supported by the Federal Government of Germany and the State of Thuringia.
References (38)
Unstructural biology coming of age
Curr. Opin. Struct. Biol.
(2011)Intrinsically disordered proteins: a 10-year recap
Trends Biochem. Sci.
(2012)Binding of cysteine thiolate to the Fe(III) heme complex is critical for the function of heme sensor proteins
J. Inorg. Biochem.
(2012)- et al.
Heme binds to an intrinsically disordered region of Bach2 and alters its conformation
Arch. Biochem. Biophys.
(2015) Using chemical shift perturbation to characterise ligand binding
Prog. Nucl. Magn. Reson. Spectrosc.
(2013)- et al.
Relaxation dispersion NMR spectroscopy as a tool for detailed studies of protein folding
Biophys. J.
(2009) - et al.
13C-detected protonless NMR spectroscopy of proteins in solution
Prog. Nucl. Magn. Reson. Spectrosc.
(2006) - et al.
Cystathionine beta-synthase: structure, function, regulation, and location of homocystinuria-causing mutations
J. Biol. Chem.
(2004) - et al.
Redox regulation and reaction mechanism of human cystathionine-beta-synthase: a PLP-dependent hemesensor protein
Arch. Biochem. Biophys.
(2005) - et al.
A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants
J. Magn. Reson.
(1982)
Novel 13C direct detection experiments, including extension to the third dimension, to perform the complete assignment of proteins
J. Magn. Reson.
Toward optimal-resolution NMR of intrinsically disordered proteins
J. Magn. Reson.
Protein conformational disorder and enzyme catalysis
Top. Curr. Chem.
Intrinsically disordered proteins in the neurodegenerative processes: formation of tau protein paired helical filaments and their analysis
Cell. Mol. Neurobiol.
Unfoldomics of human diseases: linking protein intrinsic disorder with diseases
BMC Genom.
Heme impairs the ball-and-chain inactivation of potassium channels
Proc. Natl. Acad. Sci. U. S. A.
A heme-binding domain controls regulation of ATP-dependent potassium channels
Proc. Natl. Acad. Sci. U. S. A.
Charge-state-distribution analysis of Bach2 intrinsically disordered heme binding region
J. Biochem.
Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli
BBA
Cited by (4)
The multifaceted roles of sulfane sulfur species in cancer-associated processes
2021, Biochimica et Biophysica Acta - BioenergeticsCitation Excerpt :Moreover, CBS activity is sensitive to the redox environment via i) the non-catalytic b-type heme cofactor that in its reduced form can bind the gaseous inhibitors CO and NO [28–34] and ii) a redox active disulfide bond (Cys272-Cys275) in a CXXC motif located in the catalytic domain [35]. The function of an additional heme-binding site located at the N-terminal domain remains to be elucidated [36,37]. Differently from CBS, CSE activity appears to be primarily regulated at the transcriptional level, protein expression being highly inducible by different stimuli, such as oxidative and endoplasmic reticulum stress.
<sup>13</sup>C Direct Detected NMR for Challenging Systems
2022, Chemical Reviews