The NMR contribution to protein-protein networking in Fe-S protein maturation

Abstract

Iron-sulfur proteins were among the first class of metalloproteins that were actively studied using NMR spectroscopy tailored to paramagnetic systems. The hyperfine shifts, their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues are an efficient fingerprint of the nature and the oxidation state of the Fe-S cluster. NMR significantly contributed to the analysis of the magnetic coupling patterns and to the understanding of the electronic structure occurring in [2Fe-2S], [3Fe-4S] and [4Fe-4S] clusters bound to proteins. After the first NMR structure of a paramagnetic protein was obtained for the reduced E. halophila HiPIP I, many NMR structures were determined for several Fe-S proteins in different oxidation states. It was found that differences in chemical shifts, in patterns of unobserved residues, in internal mobility and in thermodynamic stability are suitable data to map subtle changes between the two different oxidation states of the protein. Recently, the interaction networks responsible for maturing human mitochondrial and cytosolic Fe-S proteins have been largely characterized by combining solution NMR standard experiments with those tailored to paramagnetic systems. We show here the contribution of solution NMR in providing a detailed molecular view of "Fe-S interactomics". This contribution was particularly effective when protein-protein interactions are weak and transient, and thus difficult to be characterized at high resolution with other methodologies.

Keywords: CIA machinery; Fe–S proteins; Hyperfine interactions; ISC machinery; Interactomics; NMR spectroscopy.

Fig. 1

1D ¹H NMR spectra of different Fe–S cluster types. 400 MHz 1D ¹H NMR spectra of Fe³⁺ (a) and Fe²⁺ (b) C. pasteurianum rubredoxin, acquired at 308 K (adapted from [13]); c 200 MHz 1D ¹H NMR spectrum of [2Fe–2S]²⁺ P. umbilicalis ferredoxin, acquired at 303 K [15]; d 360 MHz 1D ¹H NMR spectrum of [2Fe–2S]⁺ P. umbilicalis ferredoxin, recorded at 303 K [25]; e 400 MHz 1D ¹H NMR spectrum of [2Fe–2S]⁺ human ferredoxin, acquired at 303 K [201]; f 500 MHz 1D ¹H NMR spectrum of [3Fe–4S]⁺ P. furiosus ferredoxin, recorded at 303 K [33]; 600 MHz 1D ¹H NMR spectra of [4Fe–4S]²⁺, g [40] and [4Fe–4S]³⁺, i [60] E. halophila HIPIP II, recorded at 300 K; h 600 MHz 1D ¹H NMR spectrum of [4Fe–4S]⁺ C. acidi urici ferredoxin, acquired at 298 K [43]

Fig. 2

Magnetic coupling and electronic distribution in [3Fe–4S] and [4Fe–4S] clusters. Schematic representation of the spin frustration in a [3Fe–4S] cluster (a) and of the coupling scheme in a [4Fe–4S] cluster (b). c–e Electronic distribution in the [4Fe–4S]³⁺ clusters of HiPIPs: c the extra electron can be unevenly distributed among the iron ions Fe₁, Fe₃ and Fe₄; d a chemical equilibrium between two different electronic distributions in the cluster, where mixed-valence Fe^2.5+–Fe^2.5+ iron ion pairs are represented as grey squares and purely Fe³⁺–Fe³⁺ pairs are represented as white squares; e illustration of the resonance between two limit formulas. Fe³⁺ and Fe²⁺ ions are represented as white and black squares, respectively

Fig. 3

The ¹H–¹⁵N IR-HSQC-AP NMR experiment: a new tool for paramagnetic Fe–S proteins. Schematic drawings of the pulse sequence of the ¹H–¹⁵N IR-HSQC-AP NMR experiment. The inversion recovery delay τ and the coherence transfer delay δ must be chosen according to, respectively, T₁ and T₂ relaxation properties of the signals of interest. b Efficiency of an INEPT transfer function at different ¹H T₂ values: b 100 ms, c 10 ms, d 5 ms, e 2 ms, f 1 ms, g 0.5 ms. Relaxation is neglected in a. Letters have been drawn at the correspondence of the maximum values for each transfer function. A dashed line is shown at the 2.65 ms of INEPT step (94 Hz for ¹H–¹⁵N J coupling). A solid line is shown in correspondence of 10% transfer efficiency. The latter is a limit threshold below which direct excitation of ¹⁵N spins should replace the INEPT step in the first part of the sequence. c Optimized ¹H–¹⁵N IR-HSQC-AP experiment vs. d standard ¹H–¹⁵N HSQC experiment acquired on 500 MHz at 298 K on the [2Fe–2S]²⁺-CIAPIN1 domain of human anamorsin

Fig. 4

Solution NMR as a tool to investigate weak, transient protein–protein interactions in Fe–S protein maturation pathways. Weak and transient protein–protein interactions are detected by following backbone NH chemical shift changes occurring in a standard ¹H–¹⁵N HSQC NMR experiment and in a ¹H–¹⁵N IR-HSQC-AP NMR experiment, upon titrating ¹⁵N-labeled protein with the unlabeled protein partner and vice versa. Standard ¹H–¹⁵N HSQC experiments allow the identification of protein–protein interacting regions far from the paramagnetic Fe–S cluster (showed in cyano), and to estimate the dissociation constant (K_d) of the observed interaction. ¹H–¹⁵N IR-HSQC-AP NMR experiment allows to identify protein–protein interacting regions close to the paramagnetic Fe–S cluster (showed in yellow). 1D ¹H NMR experiment provides information on the kind of Fe–S cluster(s) bound or assembled on a target protein or protein–protein complex, on the redox state(s) of the cluster(s), and on the cluster ligands

Fig. 5

The NMR contribution to the investigation of [4Fe–4S] clusters formation in the mitochondrial iron–sulfur cluster assembly machinery. On the basis of standard and paramagnetic systems-tailored NMR experiments, we provide a model for the transfer of two [2Fe–2S]²⁺ clusters from GLRX5 to ISCA1–ISCA2 hetero-dimeric complex: in solution dimeric [2Fe–2S]²⁺ GLRX5 has two states in equilibrium with each other, differing in the binding mode of the GSH molecules ([2Fe–2S] GLRX5_a and [2Fe–2S] GLRX5_b); dimeric [2Fe–2S]²⁺ GLRX5 specifically transfers the cluster to apo ISCA1–ISCA2 hetero-dimeric complex via an associative process that involves a transient protein–protein intermediate; [2Fe–2S] GLRX5_b is more reactive than [2Fe–2S] GLRX5_a to donate the cluster; ISCA1–ISCA2 hetero-dimeric complex is obtained in solution by exchanging one subunit of the ISCA2 symmetric dimer with one subunit of ISCA1, which, as isolated protein, is present in solution in a monomer–dimer equilibrium; the two [2Fe–2S]²⁺ clusters received preferentially by [2Fe–2S] GLRX5_b are reductively coupled on ISCA1–ISCA2 hetero-dimeric complex to form a [4Fe–4S]²⁺ cluster

Fig. 6

The NMR contribution to the elucidation of  the GLRX3-dependent anamorsin maturation pathway. Standard ¹H–¹⁵N-HSQC and ¹H–¹⁵N IR-HSQC-AP NMR experiments, combined with UV–Vis and EPR spectroscopy, showed that GLRX3 forms a 1:1 hetero-dimeric complex with anamorsin, in which both clusters from [2Fe–2S]₂ GLRX3₂ are transferred to anamorsin. In its mature holo state anamorsin interacts with NDOR1, forming a specific protein complex, where the anamorsin unstructured linker tightly interact with NDOR1, while the C-terminal CIAPIN1 domain of anamorsin, containing the [2Fe–2S] redox center, only transiently interacts, through complementary charged residues, with the FMN-binding domain of NDOR1 to perform the electron transfer reaction. Standard NMR experiments showed that the [2Fe–2S]₂ GLRX3–BOLA2₂ hetero-complex transfers in vitro both [2Fe–2S]²⁺ clusters to apo anamorsin, producing its mature holo state, and that this process goes via the same protein–protein recognition mechanism operating in the GLRX3-anamorsin interaction, i.e., specifically occurring between the N-terminal domains of the two proteins. The BOLA2–GLRX3 complex might be released in solution upon the interaction of holo anamorsin with NDOR1