Structural biology study of human TNF receptor associated factor 4 TRAF domain

TRAF4 is a unique member of TRAF family, which is essential for innate immune response, nervous system and other systems. In addition to be an adaptor protein, TRAF4 was identified as a regulator protein in recent studies. We have determined the crystal structure of TRAF domain of TRAF4 (residues 292-466) at 2.60 Å resolution by X-ray crystallography method. The trimericly assembled TRAF4 resembles a mushroom shape, containing a super helical "stalk" which is made of three right-handed intertwined α helixes and a C-terminal "cap", which is divided at residue L302 as a boundary. Similar to other TRAFs, both intermolecular hydrophobic interaction in super helical "stalk" and hydrogen bonds in "cap" regions contribute directly to the formation of TRAF4 trimer. However, differing from other TRAFs, there is an additional flexible loop (residues 421-426), which contains a previously identified phosphorylated site S426 exposing on the surface. This S426 was reported to be phosphorylated by IKKα which is the pre-requisite for TRAF4-NOD2 complex formation and thus to inhibit NOD2-induced NF-κB activation. Therefore, the crystal structure of TRAF4-TRAF is valuable for understanding its molecular basis for its special function and provides structural information for further studies.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25°C in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40 percent dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).