Dissection of the functional and structural domains of phosphorelay histidine kinase A of Bacillus subtilis

Abstract

The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.

FIG. 1

Domain structure of kinase A and extent of fragments used to probe kinase A activities. The top diagram shows the location and extent of the PAS domains (PAS-A, PAS-B, and PAS-C), the histidine phosphotransfer domain (His), and the ATP-binding domain in the primary sequence of kinase A. All numbers indicate the relevant amino acids where fragments start or stop. The KinA-N, KinA-M, and KinA-C fragments delineate the extent of the constructs expressed as proteins that were originally defined by the protease V8 cleavage sites of intact kinase A. MBP fragment numbers indicate the N-terminal amino acid of kinase A fused in frame to the MBP. N fragment numbers indicate the C-terminal amino acid of the kinase A fragment used in the yeast two-hybrid system.

FIG. 2

Molecular weight determination of expressed KinA domains by gel filtration. The molecular weights of expressed KinA fragments were analyzed using a Sephacryl S-100 FPLC column. Standard molecular weight markers (⧫) were bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4 kDA), and aprotinin (6.5 kDa). The column's voided volume (V₀) was determined by the migration of dextran blue (2,000 kDa). KinA-N, KinA-M, and KinA-C were eluted in a single peak at the elution volumes (V_e) of 66, 28, and 41 ml, respectively. Their molecular sizes were determined as 13, 60, and 73.3 kDa.

FIG. 3

Initial rates of autophosphorylation of MBP-kinase A fusion proteins. Autophosphorylation from [γ-³²P]ATP was analyzed as a function of time. Samples were separated on SDS-PAGE and labeled KinA was quantitated by phosphorimaging. Symbols: ○, kinase A; ▵, MBP-1 fusion; ▿, MBP-13 fusion; □, MBP-101 fusion; ◊, MBP-143 fusion.

FIG. 4

Phosphorylation activity of KinA and KinA-C. Various combinations of KinA (0.5 μM), KinA-C (5 μM), and Spo0F (10 μM), as indicated, were incubated with [γ-³²P]ATP at 25°C for either 3 min or 1 h, as indicated. The phosphorylated proteins were subjected to SDS-PAGE and autoradiography as described in Materials and Methods.

FIG. 5

Phosphotransfer reactions of KinA, KinA-C, and NRII. Various combinations of KinA (0.5 μM), KinA-C (5 μM), Spo0F (10 μM), NRII (0.5 μM), and NRI (5 μM) were incubated with [γ-³²P]ATP at 25°C for 3 min. Individual reaction contents are indicated above the gel.

FIG. 6

Phosphotransfer from purified Spo0F∼P to KinA and KinA-C. Purified γ-³²P-labeled, phosphorylated Spo0F∼P (10 pmol) was incubated with various combinations of KinA-C (1 μM) and KinA (1 μM), as indicated, in reaction buffer at 25°C for 3 min. The samples were subjected to SDS-PAGE and autoradiography as described in Materials and Methods.

FIG. 7

Phosphotransfer from KinA∼P and KinA-C∼P to ADP. Purified γ-³²P-labeled KinA∼P (10 pmol) or KinA-C∼P (10 pmol) was incubated in the presence or absence of ADP (400 μM) as indicated in reaction buffer at 25°C for 3 min. The samples were subjected to TLC and autoradiography as described in Materials and Methods.

FIG. 8

UV cross-linking of [γ-³²P]ATP and [α-³²P]ATP to the KinA carboxy-terminal domain. KinA-C (5 μM) was incubated for 30 min on ice with 80 μCi of [γ-³²P]ATP (lanes 1 and 2) or [α-³²P]ATP (lanes 3 and 4) at a final concentration of 2 μM total ATP. Samples were exposed to UV light (lanes 2 and 4) or kept in the dark (lanes 1 and 3) for 60 min on ice and then subjected to SDS-PAGE and autoradiography.