How 'arm-twisting' by the inducer triggers activation of the MalT transcription factor, a typical signal transduction ATPase with numerous domains (STAND).
By characterizing this locked variant, I found that the inducer is recognized in two steps: it first binds to the sole sensor with low affinity, which then triggers the recruitment of the arm to form a high-affinity arm-sensor inducer-binding site.Through this toggling between two mutually exclusive states reminiscent of a single-pole double-throw switch, the arm couples inducer binding to NOD opening, shown here to precede nucleotide exchange.This scenario likely holds for other STANDs like mammalian NLR innate immunity receptors.
Affiliation: Institut Pasteur, Molecular Genetics Unit, Microbiology Department, F-75015 Paris, France CNRS, ERL3526, F-75015 Paris, France firstname.lastname@example.org.
- DNA-Binding Proteins/chemistry*/genetics/metabolism*
- Escherichia coli Proteins/chemistry*/genetics/metabolism*
- Transcription Factors/chemistry*/genetics/metabolism*
- Adenosine Triphosphatases/chemistry/genetics/metabolism
- Amino Acid Sequence
- Amino Acid Substitution
- Base Sequence
- Escherichia coli/genetics/metabolism
- Models, Molecular
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Protein Conformation
- Protein Interaction Domains and Motifs
- Signal Transduction
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Figure 1: Residue 70 of the MalT NBD can be cross-linked with residues of the arm domain. (A) Schematic representation of the MalT primary sequence. NBD, nucleotide binding domain; HD helical domain; WHD, winged-helix domain; DBD, DNA-binding effector domain. In the upper part of the cartoon, the location of residue Q70 and the limits of the peptides (black bars and dotted lines) that were found cross-linked to AET-derivatized C70 after irradiation of HMalTC-,Q70C in the resting form, followed by endoproteinase LysC or trypsin digestion (see text), are indicated. NODh and ASh indicate the proteinase K sensitive hinges (34). (B) The STAND activation scheme of MalT (same color coding). The transition from the resting to the multimerization competent form involves three events: inducer binding to the sensor, NOD isomerization, nucleotide exchange. The precise sequence of these events is not known, and the role of the sensor and the arm in this process is unclear (indicated by dotted outlines). (C) Model of the MalT structure from aa 1 to 417. Color coding is as in (A) except for Q70 and E395, which are represented in green. ADP is colored according to its atoms. (D) Analysis of endoproteinase LysC proteolysis products of AET-cross-linked HMalTC-,Q70C (See also Fig. S1B, C, D). Endoproteinase LysC digests of 4 µg HMalTC-,Q70C were analyzed by SDS-PAGE in the presence (+) or absence (-) of DTT. Molecular weight of markers are indicated in kDa, 18 kDa and 14 kDa fragments are highlighted. (E) HMalTQ70C,E395C disulfide bond formation detected by SDS-PAGE. Proteins HMalT, HMalTQ70C, HMalTE395C and HMalTQ70C,E395C were analyzed by SDS-PAGE in the presence and absence of DTT. The + signs indicate the subsitution(s) harboured by the protein (Q70C, E395C), whether the protein was pretreated with DTT as a first step (DTT pretreatment, see Supplementary Materials and Methods), whether the sample was treated with N-ethylmaleimide (NEM) and whether DTT was present during SDS-PAGE analysis (DTT).
STAND are large, multidomain proteins built according to a conserved scheme (2). Their hallmark is the nucleotide-binding oligomerization domain (NOD) which is basically the nucleotide binding domain-helical domain (NBD-HD) moiety of the AAA+ ATPases supplemented with a winged-helix domain (WHD) at its C-terminus (Figure 1A). A non-conserved sensor domain, which binds the inducer, is connected to the C-terminus of the NOD, either directly or through a helical domain called the arm (or HD2, or SH). The sensor domain is generally composed of repeated units like the leucine-rich (LRR), tetratricopeptide (TPR), WD40 or ankyrin repeat. At either end of the protein, one or several effector domains (e.g. protein recruitment, enzymatic or nucleic acid binding domains) are responsible for downstream signaling.