Substrate recognition and ATPase activity of the E. coli cysteine/cystine ABC transporter YecSC-FliY

Abstract

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A

“…Adding the term for the Hill coefficient yielded an nHILL=1.08, indicating that the two ATP binding sites of baMntBC do not hydrolyze ATP cooperatively. This differs from the cooperative ATP hydrolysis that was reported for both type-I and type -II ABC importers (e.g., MetNI, HisPQM, MalFGK, J o u r n a l P r e -p r o o f YecSC, and BtuCD) (34,37,62,64,65). Notably, even in a membrane environment baMntBC has very high rates of uncoupled (absence of substrate and SBP) ATPase activity (kcat = 1.85 s -1 ), similar to the SBP:substrate-stimulated ATPase rates reported for type-I importers (57,62).…”

“…We then repeated these experiments incorporating also manganese into the vesicles' lumen at a 50:10:1 manganese: baMntA: baMntBC molar ratio, and observed a similarly modest inhibitory effect (Figure 6A). Collectively, these results suggest that similar to the type-II ABC importer BtuCD yet unlike the Type-I ABC importers MalFGK, YecSC and HisPQM baMntBC has high basal ATPase activity that is not further stimulated by the SBP:substrate complex (34,37,57,58,62).…”

Characterization of the ABC methionine transporter fromNeisseria meningitidisreveals that MetQ is a lipoprotein

“…Maximal stimulation by liganded-SBPs is also a mechanistic feature shared by the ABC importers EcMalFGK 2 ( Davidson et al, 1992 ) and EcHisQMP 2 ( Ames et al, 1996 ). In contrast, for the ABC importer EcYecSC-FliY, full stimulation of ATPase can be achieved in both the liganded-SBP and the unliganded-SBP ( Sabrialabed et al, 2020 ). Although the origin of these differences are unclear, our data show that NmMetNI is tightly coupled and highlight the mechanistic differences between ABC importers.…”

Escherichia coli K-12 Lacks a High-Affinity Assimilatory Cysteine Importer

“…As this paper was being prepared for publication, Oded Lewinson communicated to us that his group had observed binding of cysteine in vitro to E. coli TcyJ, the periplasmic binding protein of the ABC-type cystine importer, and that this binding stimulated ATP hydrolysis by the associated TcyLN components ( 57 ). Binding at this site is surprising, because the side chain of cysteine (-CH 2 -SH) does not resemble the effective side chain of cystine (-CH 2 -S-S-CH 2 -CH-[COO-]-NH 3 + ).…”