Abstract
Escherichia coli HisJ is a type II periplasmic binding protein that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here, we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2, we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, whereas D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, whereas isolated D2 experiences a significant reduction in slower (millisecond to microsecond) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact with each other in the presence or absence of histidine, which indicates the importance of intact interdomain-connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process.
Highlights
HisJ is a bilobal periplasmic binding protein that mediates basic amino acid transport
HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain
Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process
Summary
HisJ is a bilobal periplasmic binding protein that mediates basic amino acid transport. The apo-forms of type II PBPs (e.g. LAOBP- and glutamine-binding protein) (14 –16) have been crystallized in an open conformation wherein there are few interdomain contacts except for regions adjacent to the hinge regions. For these PBPs, ligand binding occurs in the interdomain cleft region and is accompanied by significant closing and twisting motions that bring the domains together [14, 17]. Our results support the idea that the individual domains of HisJ retain the same global fold as in the WT protein; there are apparent functional differences related to the absence of an intact hinge region. These results strongly support the idea that ligand binding is initiated by the D1 domain of HisJ and that the intact linker region is required for transmission of the “closing” signal to the D2 region
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