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The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra β-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

Numerous biomolecules possess α-D-glucosamine as structural component. However, chemical glycosylations aimed at this backbone are usually not easily attained without generating the unwanted β-isomer. We report herein a versatile approach in affording full α-stereoselectivity built upon a carefully selected set of orthogonal protecting groups on a D-glucosaminyl donor. The excellent stereoselectivity provided by the protecting group combination was found independent of leaving groups and activators. With the trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan, heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was found to bind at least six sugar units with the interaction notably being entropically driven.