Crystal structures of inactive PAK1(K299R) and the activation (A)-loop phospho-mimetic PAK1(T423E) have suggested that the kinase domain is in an active state regardless of activation loop status. Contrary to a large body of literature, we find that neither is PAK1(T423E) active in cells, nor does it exhibit significant activity in vitro. To explain these discrepancies all-atom molecular dynamics (MD) simulations of PAK1(phospho-T423) in complex with ATP and substrate were performed. These simulations point to a key interaction between PAK1 Lys308, at the end of the alphaC helix, and the pThr423 phosphate group, not seen in X-ray structures. The orthologous PAK4 Arg359 fulfills the same role in immobilizing the alphaC helix. These in silico predictions were validated by experimental mutagenesis of PAK1 and PAK4. The simulations explain why the PAK1 A-loop phospho-mimetic is inactive, but also point to a key functional interaction likely found in other protein kinases.
The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.
The mechanism of coupling of ion pumping in the membrane-bound A(O) sector with ATP synthesis in the A(3)B(3) headpiece of the A(1) sector in the A(1)A(O) ATP synthase is a puzzle. Previously, crosstalk between the stalk and nucleotide-binding subunits F(Mm) and B(Mm) of the Methanosarcina mazei Gö1 A-ATP synthase has been observed by nucleotide-dependent cross-link formation of both subunits inside the enzyme. The recently determined NMR solution structure of F(Mm) depicts the protein as a two-domain structure, with a well-folded N-terminus having 78 residues and a flexible C-terminal part (residues 79-101), proposed to become structured after binding to its partner, B(Mm). Here, we detail the crucial interactions between subunits B(Mm) and F(Mm) by determining the NMR structure of the very C-terminus of F(Mm), consisting of 20 residues and hereafter termed F(Mm(81-101)), and performing molecular dynamics simulations on the resulting structure. These data demonstrate that the flexibility of the C-terminus enables F(Mm) to switch between an elongated and retracted state. Docking and MD in conjunction with previously conducted and published NMR results, biochemical cross-linking, and fluorescence spectroscopy data were used to reconstruct a model of a B(Mm)-F(Mm) assembly. The model of the B(Mm)-F(Mm) complex shows the detailed interactions of helices 1 and 2 of the C-terminal domain of B(Mm) with the C-terminal residues of F(Mm). Movements of both helices of B(Mm) accommodate the incoming C-terminus of F(Mm) and connect the events of ion pumping and nucleotide binding in the A(1)A(O) ATP synthase.
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