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X-ray structures of NS1 effector domain mutants.

ABSTRACT: The influenza A virus nonstructural protein NS1 is a multifunctional dimeric protein that acts as a potent inhibitor of the host cellular antiviral state. The C-terminal effector domain of NS1 binds host proteins, including CPSF30, and is a target for the development of new antiviral drugs. Here we present crystallographic structures of two mutant effector domains, W187Y and W187A, of influenza A/Udorn/72 virus. Unlike wild-type, the mutants behave exclusively as monomers in solution based on gel filtration data and light scattering. The W187Y mutant is able to bind CPSF30 with a binding affinity close to the wild-type protein; that is, it retains a receptor site for aromatic ligands nearly identical to the wild-type. Therefore, this monomeric mutant protein could serve as a drug target for a high throughput inhibitor screening assays, since its binding pocket is unoccupied in solution and potentially more accessible to small molecule ligands.

SUBMITTER: Xia S

PROVIDER: S-EPMC2819631 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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X-ray structures of NS1 effector domain mutants.

Xia Shuangluo S Robertus Jon D JD

Archives of biochemistry and biophysics 20091206 2

The influenza A virus nonstructural protein NS1 is a multifunctional dimeric protein that acts as a potent inhibitor of the host cellular antiviral state. The C-terminal effector domain of NS1 binds host proteins, including CPSF30, and is a target for the development of new antiviral drugs. Here we present crystallographic structures of two mutant effector domains, W187Y and W187A, of influenza A/Udorn/72 virus. Unlike wild-type, the mutants behave exclusively as monomers in solution based on ge ...[more]

PMID: 19995550

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Project description:The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg(2+) bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerization of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving alpha1, loop beta5alpha5 and N terminus of alpha5 elements, also seen in the complex of PhoB RD with Mg(2+), in which helix alpha4 highly increases its flexibility. PhoB RD in complex with Mg(2+) and BeF(3) (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix alpha4 and shows another interaction involving alpha4, beta5 and alpha5 segments. We have selected a series of constitutively active PhoB mutants (PhoB(CA)) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoB(CA) RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix alpha4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein.

Dataset Information

X-ray structures of NS1 effector domain mutants.

Publications

X-ray structures of NS1 effector domain mutants.

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