An adaptor for feedback regulation of heme biosynthesis by the mitochondrial protease CLPXP.

Heme biosynthesis is tightly coordinated such that essential heme functions including oxygen transport, respiration, and catalysis are fully supplied without overproducing toxic heme precursors and depleting cellular iron. The initial heme biosynthetic enzyme, ALA synthase (ALAS), exhibits heme-induced degradation that is dependent on the mitochondrial AAA+ protease complex CLPXP, but the mechanism for this negative feedback regulation had not been elucidated. By biochemical reconstitution, we have discovered that POLDIP2 serves as a heme-sensing adaptor protein to deliver ALAS for degradation. Similarly, loss of POLDIP2 strongly impairs ALAS turnover in cells. POLDIP2 directly recognizes heme-bound ALAS to drive assembly of the degradation complex. The C-terminal element of ALAS, truncation of which leads to a form of porphyria (XLDPP), is dispensable for interaction with POLDIP2 but necessary for degradation. Our findings establish the molecular basis for heme-induced degradation of ALAS by CLPXP, establish POLDIP2 as a substrate adaptor for CLPXP, and provide mechanistic insight into two forms of erythropoietic protoporphyria linked to CLPX and ALAS.

The structural basis for monoclonal antibody 5D2 binding to the tryptophan-rich loop of lipoprotein lipase.

For three decades, the LPL-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

Structural characterization of the α-N-acetylglucosaminidase, a key enzyme in the pathogenesis of Sanfilippo syndrome B.

Mucopolysaccharidosis III B (MPS III-B) is a rare lysosomal storage disorder caused by deficiencies in Alpha-N-acetylglucosaminidase (NAGLU) for which there is currently no cure, and present treatment is largely supportive. Understanding the structure of NAGLU may allow for identification of novel therapeutic targets for MPS III-B. Here we describe the first crystal structure of human NAGLU, determined to a resolution of 2.3 Å. The crystal structure reveals a novel homotrimeric configuration, maintained primarily by hydrophobic and electrostatic interactions via domain II of three contiguous domains from the N- to C-terminus. The active site cleft is located between domains II and III. Catalytic glutamate residues, E316 and E446, are located at the top of the (α/ß)8 barrel structure in domain II. We utilized the three-dimensional structure of NAGLU to map several MPS III-B mutations, and hypothesize their functional consequences. Revealing atomic level structural information about this critical lysosomal enzyme paves the way for the design of novel therapeutics to target the underlying causes of MPS III-B.