The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly

Abstract

Background: Assimilatory NADPH-sulfite reductase (SiR) is an essential metalloenzyme for sulfur metabolism made from two subunits. Results: We defined how the subunits of SiR assemble, with or without cofactors. Conclusion: One region of the metalloenzyme interacted either with its reductase partner when cofactors were formed or with itself when they were not. Significance: We propose a novel mechanism to regulate SiR assembly.

Small-angle neutron scattering solution structures of NADPH-dependent sulfite reductase

“…An additional variant consists solely of its C-terminal, 43 kDa FNR/connection domains (Covès et al , 1999; Zeghouf et al ., 2000, SiRFP-43), representing a minimal, inactive version of SiRFP (Figure 1C). Full-length SiRHP was used in all studies because when its N-terminus is removed it can no longer bind SiRFP (Askenasy et al ., 2015); however, those amino acids are not present in the X-ray crystal structure (Crane et al , 1995, Figure 1B).…”

“…Although structures of the monomeric SiR subunits are known from X-ray crystallography (Crane et al ., 1995; Gruez et al ., 2000; Tavolieri et al ., 2019), no structures exist for higher-order complexes. Consequently, we do not know how the subunits interact such that there is a tight-binding, structural interface independent of the transient, functional interface that mediates electron transfer (Askenasy et al ., 2018; Askenasy et al ., 2015). Additionally, we do not know if subunit assembly affects the relative domain orientations of the conformationally dynamic SiRFP.…”

“…In our model for the positions of SiRFP-60 and SiRHP within the envelopes modeled from neutron scattering, SiRHP is far from the Fld domain that would be responsible for funneling electrons to it within this minimal dimer (Figure 3D). This placement is consistent with the results from contrast matching experiments (Figures 4 and 5) as well as previously-reported mutational analysis that identified amino acids in the FNR domain of SiRFP as important for SiRHP binding (Askenasy et al ., 2018; Askenasy et al ., 2015). Nevertheless, cis electron transfer to a tightly-bound SiRHP in the minimal dimeric complex occurs, albeit at a rate of ∼50% that of the holoenzyme (Tavolieri et al ., 2019; Zeghouf et al ., 2000).…”

“…Another aspect of SiR that sets it apart from analogous systems is the way in which SiRFP and SiRHP interact, through interactions between SiRHP’s N-terminus and a position on SiRFP’s FNR domain that is far from the Fld domain (Askenasy et al , 2018; Askenasy et al , 2015). This interaction is counterintuitive because the Fld domain must interact with SiRHP to pass electrons to its siroheme-Fe 4 S 4 cofactors that funnel the electrons to the siroheme-bound substrate.…”

Mapping the contact surfaces in the Lamin A:AIMP3 complex by hydrogen/deuterium exchange FT-ICR mass spectrometry

“…In addition to X-ray crystallography, other approaches for study of protein-ligand and protein-protein interaction include: cryo-electron microscopy [ 19 ], nuclear magnetic resonance (NMR) [ 20 ], electron microscopy [ 21 , 22 ], small angle X-ray scattering [ 23 ], hydrogen-deuterium exchange [ 24 , 25 ], chimeric molecule analysis [ 26 ], mutagenesis, and chemical cross-linking [ 27 ]. Although X-ray crystallography remains the most prominent and reliable method, it is not readily applicable for the AIMP3-LmnA complex, due to the highly extended and dynamic structure of LmnA (only a partial crystal structure is available for the LmnA) [ 28 ].…”