(i) Sensor Domain to Catalytic Domain
For a sensor HK to conduct its catalytic functions, a mechanism must exist by which the periplasmic loop and transmembrane domains can sense and transduce a signal to the catalytic core. Conformational changes must occur from the sensor loop to the kinase core, to transduce the signal. As stated in section 1.5.3 a number of sensory domains e.g. PAS and GAF domains are involved in sensing signals, which are believed to allow signal
transduction through their structural plasticity and flexibility. An additional domain involved in signal transmission is a HAMP domain (present in Histidine kinases, Adenyl cyclases, Methyl-accepting chemotaxis proteins and Phosphatases) (Szurmant et al., 2007).
N N C C α10 α8 α7 α9
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HAMP domains connect the last transmembrane helix of the sensor domain to the catalytic domain. They consist of two helices which form a parallel four-helical coiled coil in the homodimeric active state, and are proposed to control signal transmission to the catalytic core (Hulko et al., 2006; Ferris et al., 2012) (see Figure 1.5.5.1).
Figure 1.5.5.1: Schematic of histidine kinase modular architecture. Adapted from Szurmant et al., (2007).
Two possible models exist for the function of the HAMP domains: a cog-like rotation of each helix relative to one another (‘gearbox model’) (Hulko et al., 2006) or a combination of vertical movement and helical rotation (‘screw-like motion’) (Airola et al., 2010). The gearbox model was proposed from studies of the first HAMP structure, of Af1503, a receptor from the archaeon Archaeoglobus fulgidus. Crystal structures of this receptor indicate that an axial rotation of the four-helix bundle by 26o occurs in the active state to transmit a signal to the kinase domain (Hulko et al., 2006). This rotation converts the domain packing from a ‘complementary x-da’ packing to a ‘knobs-into-holes’ geometry.
C Periplasmic Sensing Transmembrane Helices HAMP Domain Cytoplasmic Sensing) Catalytic Core
(DHp & CA) ATP ATP
C N N Sen sor c omp le x
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The screw-like signalling mechanism is based on the crystal structure of the receptor Aer2 from P. aeruginosa, which has three connected HAMP domains, each containing a parallel four-helix bundle. However, in middle of HAMP2, there is an offset in the helical register between the first and second helix, which leads to a combination of vertical and helical rotation to transduce signals, i.e. a screw-like motion (Airola et al., 2010). Therefore it is likely that structural changes in the sensor region upon ligand binding could trigger rotations of the TM helices and HAMP domain, or structural changes in PAS/GAF or other domains, to transduce signals to the kinase domain (Cheung & Hendrickson, (2010), Zhang & Hendrickson, (2010).
(ii) Catalytic Domain to Dimerisation Domain
Downstream structural changes in the dimerisation domain induced by signalling elements such as the HAMP domains are likely to alter the regulation of interactions between the DHp and CA domains. The changes triggered by different HAMP conformations do not appear to perturb the active-site histidine or the lower half of the DHp domain (Wang, 2012), but the CA domain must rotate to transfer a phosphate to the active His site in the DHp domain. As stated in section 1.5.3, numerous atomic structures exist for the isolated cytoplasmic domains of different HKs including EnvZ (Tomomori et al., 1999), PhoQ (Marina et al., 2001) and HK853 (Marina et al., 2005). The crystal structure of HK853 from T. maritima is
particularly useful for discussion since a recent co-crystal structure of the kinase and its cognate response regulator RR468 has been published (Casino et al., 2009). The crystal structure of the entire cytoplasmic portion of HK853 is shown in Figure 1.5.5.2, which represents a dimeric conformational state ready for phosphotransfer (Marina et al., 2005).
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Figure 1.5.5.2: Ribbon diagram of the dimeric cytoplasmic portion of HK853. Adapted from Casino et al., 2009. Each subunit is coloured in pale blue or light brown, with the H260 site and bound phosphate mimic (SO4
2-
) shown as spheres, drawn in PyMol (PDB: 2C2A).
The CA domain characteristically hosts one ATP molecule between its ATP lid and a central helix, which can be exposed for attack. In this instance the structure contains an ADPβN, instead of ATP. The CA domain is also connected to the DHp domain via a mobile hinge, which has been observed in other HKs to adopt multiple conformations for phosphotransfer. The H260 side chain is exposed to the surface of the protein and there is a sulphate ion in the structure bound to H260, mimicking the phosphorylated histidine.
As HKs are homodimeric, there are two mobile phosphorylating domains (CA) and two phospho-accepting exposed His residues (DHp). Therefore cis- or trans-phosphorylation could occur. Cis-phosphorylation is the phosphorylation of one HK monomer by itself, whereas trans-phosphorylation is phosphorylation of one HK monomer by the other. In the HK853 structure, the His residue and ATP binding site are 19Å apart, requiring a rotation of domains of 70o for trans- phosphorylation (Marina et al., 2005). This is not consistent with 26o rotations observed in HAMP domains, so cis-phosphorylation is the preferred model.
DHp CA ADPβN H260 N α1 α2 SO42-
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However, there has been debate over which model applies, since e.g. EnvZ and DesK (Tomomori et al., 1999; Trajtenberg et al., 2010) autophosphorylate in a trans-mechanism, whereas HK853 and PhoR (Casino et al., 2009; 2010) autophosphorylate in cis. In fact, a recent paper has demonstrated that the mechanism of autophosphorylation is independent of the cis- or trans-directionality since the catalytic centre of HK853 and EnvZ appear identical in functional and structural data (Casino et al., 2014), and that the directionality is dependent on the left or right handedness of the DHp α1 and α2 helices.
(iii) Dimerisation Domain to Response Regulator
The crystal structure of the HK853-RR468 complex is shown in Figure 1.5.5.3, and shows that the response regulator molecules are positioned on opposite sides of the DHp domain dimer and sit below the phosphorylatable His260 residue.
Figure 1.5.5.3: Ribbon representation of the crystal structure of the HK853-RR468 complex. Adapted from Casino et al., 2009.The complex is viewed perpendicular to the DHp axis, and one RR molecule is shown in the structure for clarity. Each subunit of the HK is coloured in blue or light brown, and the RR is in green.
DHp CA ADPβN H260 RR α1 α2 D53
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In this orientation the Asp53 site of the RR is aligned with a phosphoryl mimic and the H260 site of the HK (shown by spheres), favouring phosphoryl transfer (Casino et al., 2009). The RR clings to the helical stem of one HK subunit by inserting the α1 helix of the RR next to the two helices of the HK, forming a six-helix bundle (Wang, 2012).
Overall the crystal structures of HK853 with and without bound RR, show conformational changes in accordance with the different functional states (phosphorylated by ATP, and phosphotransfer to the RR). These contacts should vary with the reaction occurring and confer specificity between the HK and its cognate RR. Similar interactions are observed for the catalytic domains of other histidine kinases with their cognate response regulators, e.g. in the Spo0B-Spo0F complex in B. subtilis (Zapf et al., 2000) and in the ThkA-TrrA complex in T. maritima (Yamada et al., 2009). Based on the HK853-RR468 complex and previous structures, Casino et al., (2009), propose a model for TCS signalling (see Figure 1.5.5.4).
Figure 1.5.5.4: Signal transduction model, adapted from Casino et al., (2009). Signal transduction involves rotation of coiled coils at the top of the DHp domain, altering its packing. This causes the CA domain to either approaches the His residue, activating the kinase (centre), or moves away to generate site for unphosphorylated RR, for
phosphotransfer (right), or phosphorylated RR, triggering phosphatase function (left).
Kinase Phosphotransfer Phosphatase
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