Abstract
NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.
Highlights
Higher structural complexity, which often contain additional metals
In the case of the FeMo-co of nitrogenase, simple [Fe-S] clusters are the substrates of the NifB protein, which uses them to generate NifB-co, a complex [Fe-S] cluster with a proposed [6Fe-9S-X] structure [2,3,4]
The completed FeMo-co is incorporated into its target apoprotein, a FeMoco-deficient form of dinitrogenase, yielding an enzyme that is competent for N2 reduction
Summary
Core-NafY was shown not to bind apo-NifDK tightly [12]. There have been no reports on the structure or function of the 12-kDa N-terminal domain (residues 1–96, which we will refer to as n-NafY) or the C-terminal region of NafY (residues 231–243). We show that n-NafY is sufficient to bind apoNifDK with substantial affinity. We present the solution structure of n-NafY and characterize domain interactions in NafY by NMR. N-NafY has a sterile ␣-motif (SAM) fold, a protein-protein interaction domain that could not be inferred from its amino acid sequence. These findings strengthen the model that NafY targets apoNifDK and delivers the cofactor using two essentially independent specialized domains
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