Protein-Interaction Affinity Gradient Drives [4Fe–4S] Cluster Insertion in Human Lipoyl Synthase

Abstract

Human lipoyl synthase (LIAS) is an enzyme containing two [4Fe−4S] clusters (named FeS RS and FeS aux ) involved in the biosynthesis of the lipoyl cofactor. The mechanism by which a [4Fe−4S] cluster is inserted into LIAS has thus far remained elusive. Here we show that NFU1 and ISCA1 of the mitochondrial iron−sulfur cluster assembly machinery, via forming a heterodimeric complex, are the key factors for the insertion of a [4Fe−4S] cluster into the FeS RS site of LIAS. In this process, the crucial actor is the C-domain of NFU1, which, by exploiting a protein-interaction affinity gradient increasing from ISCA1 to LIAS, drives the cluster to its final destination.

Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins

“…While the molecular mechanism involving IND1 in the insertion of the [4Fe-4S] 2+ cluster into complex I is not yet characterized, the insertion of the [4Fe-4S] 2+ cluster into LIAS has been deeply investigated. Sequential protein–protein interactions involving ISCA1 and NFU1 have been shown to be responsible for the insertion of the [4Fe-4S] cluster into LIAS [ 32 , 33 ]. LIAS is a member of the radical S-adenosylmethionine (SAM) superfamily [ 34 , 35 ].…”

“…It catalyzes the final step of the biosynthesis of lipoyl cofactor [ 36 , 37 , 38 ] and binds two [4Fe-4S] clusters [ 39 , 40 ]: a [4Fe-4S] cluster (FeS RS ), typical of all radical SAM enzymes, and a [4Fe-4S] cluster (FeS aux ) that provides two sulfur atoms to the lipoyl cofactor [ 41 , 42 ]. It has been shown that the C-domain of NFU1 drives first [4Fe-4S] 2+ cluster delivery from the ISCA1-ISCA2 complex, where the [4Fe-4S] 2+ cluster is assembled, to a [4Fe-4S] 2+ ISCA1-NFU1 intermediate complex, which then specifically directs the cluster into the FeS RS site of LIAS [ 32 , 33 ]. According to this molecular function, NFU1 has been shown to form two hetero-complexes with ISCA1 and LIAS [ 32 , 33 ].…”

“…It has been shown that the C-domain of NFU1 drives first [4Fe-4S] 2+ cluster delivery from the ISCA1-ISCA2 complex, where the [4Fe-4S] 2+ cluster is assembled, to a [4Fe-4S] 2+ ISCA1-NFU1 intermediate complex, which then specifically directs the cluster into the FeS RS site of LIAS [ 32 , 33 ]. According to this molecular function, NFU1 has been shown to form two hetero-complexes with ISCA1 and LIAS [ 32 , 33 ]. These two complexes have been recently characterized at a molecular level, and it has been shown that, in both cases, only the C-domain of NFU1 is involved in the protein–protein interaction [ 32 , 33 ].…”

“…According to this molecular function, NFU1 has been shown to form two hetero-complexes with ISCA1 and LIAS [ 32 , 33 ]. These two complexes have been recently characterized at a molecular level, and it has been shown that, in both cases, only the C-domain of NFU1 is involved in the protein–protein interaction [ 32 , 33 ]. This domain therefore drives the assembled [4Fe-4S] 2+ cluster from the ISCA1-ISCA2 complex to the final destination.…”

“…Two Asp residues are located nearby to Arg182, and this charge loss in the pathogenic mutants can thus perturb these electrostatic interactions, destabilizing the overall structure as well as protein–protein recognition ( Figure 3 B). Arg182 is solvent exposed and is located on the helix of the C-domain of NFU1 involved in the complex formation with both ISCA1 and LIAS [ 32 , 33 ]. Its mutation into Gln or Trp might negatively affect the protein–protein recognition between the C-domain of NFU1 and both ISCA1 and LIAS and the [4Fe-4S] cluster transfer to LIAS.…”