Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis-and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.