Human UDP-α-d-xylose synthase forms a catalytically important tetramer that has not been observed in crystal structures.

Human UDP-α-d-xylose synthase (hUXS) is a member of the extended short chain dehydrogenase/reductase (SDR) family of enzymes. Previous crystallographic studies have shown that hUXS conserves the same dimeric quaternary structure observed in other SDR enzymes. Here, we present evidence that hUXS also forms a tetramer in solution that is important for activity. Sedimentation velocity studies show that two hUXS dimers undergo a concentration-dependent association to form a tetramer with a Kd of 2.9 µM. The tetrameric complex is also observed in small-angle X-ray scattering (SAXS). The specific activity for the production of the reaction intermediate UDP-α-d-4-keto-xylose displays a hyperbolic dependence on protein concentration that is well modeled by an isotherm using the 2.9 µM Kd of the tetramer. Likewise, the rate of UDP-α-d-xylose production in the presence of increasing concentrations of the small molecule crowder trimethylamine N-oxide is consistent with the formation of a higher activity tetramer. We present several possible structural models of the hUXS tetramer based on (i) hUXS crystal packing, (ii) homology modeling, or (iii) ab initio simulated annealing of dimers. We analyze the models in terms of packing quality and agreement with SAXS data. The higher activity of the tetramer coupled with the relative instability of the complex suggests that an association-dissociation mechanism may regulate hUXS activity.

Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

Structures of thermophilic and mesophilic adenylate kinases from the genus Methanococcus.

The crystal structures of adenylate kinases from the thermophile Methanococcus thermolithotrophicus and the mesophile Methanococcus voltae have been solved to resolutions of 2.8A and 2.5A, respectively. The structures of the enzymes are similar to that of the adenylate kinase from archaeal Sulfolobus acidocaldarius in many respects such as the extended central beta-sheets, the short LID domain, and the trimeric state. The analysis of unligated and AMP-bound subunits of M.voltae suggests that movements of two mobile domains are not independent of each other. The methanococcal structures are examined with respect to their lack of the "invariant" Lys residue within the phosphate-binding loop, and two Arg residues in the LID domain are proposed as substituting residues based on their conservation among archaeal adenylate kinases and mobility within the structures. Since S.acidocaldarius adenylate kinase has the invariant Lys residue as well as the two Arg residues, its phosphate-binding loop is examined and compared with those of other adenylate kinases. On the basis of the comparison and other available biochemical data, the unusual conformation of the Lys residue in S.acidocaldarius adenylate kinase is explained. Despite possessing 78% sequence identity, the methanococcal enzymes exhibit significantly different thermal stabilities. To study the determinants of thermostability, several structural features including salt-links, hydrogen bonds, packing density, surface to volume ratio and buried surface area are compared between the enzymes. From their difference in apolar buried surface area, hydrophobic interaction is proposed to be a basis for the disparate thermostabilities, and the corresponding free energy difference is also estimated. Results of previous mutational studies are interpreted in terms of the crystal structures, and support the importance of hydrophobic interactions in thermostability.