SUMMARY Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B-and C-helices and portions of the F-Ghelix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior. Cytochrome P450 (P450)4 dependent monooxygenases are involved in the biogenesis of sterols and hormones and oxidation of a broad range of xenobiotic compounds (1). Many individual mammalian cytochromes P450 can accept a wide variety of hydrophobic substrates of differing shapes and sizes and render them more hydrophilic for excretion or subsequent conjugation. In addition to their central role in drug clearance, the ability of mammalian cytochromes P450 to convert various inactive precursors to the respective bioactive compounds makes these enzymes of paramount importance for the healthcare and pharmaceutical industries (2-4).Despite their broad range of substrates, the single domain fold of P450s is well conserved across families (5-9). Hence, the ability to adapt to molecules reflects the notable plasticity of many secondary structural elements (8, 9). P450s show the ability to form compact structures around small ligands or empty active sites, as evidenced by P450 2B4 complexed with 1-(4-chlorophenyl)imidazole (1-CPI) or 4-CPI (10, 11), P450 3A4 (12), and several proteins in the 2C subfamily (13,14). Moreover, some P450s also appear able to alter their conformations to accommodate ligands of greater volume, as seen in P450 2B4 with bifonazole (15) or in P450 3A4 with erythromycin or with ketoconazole (16).Our laboratory has utilized an engineered form of P450 2B4 termed P450 2B4dH 5 (N-terminally modified and a C-terminal His tag) to gain insight into enzyme flexibility (10,11,15,(17)(18)(19). The i...

Protein-protein interactions are essential for biological function, but structures of protein-protein complexes are difficult to obtain experimentally. To derive the protein complex of the DNA-repair enzyme human uracil-DNA-glycosylase (hUNG) with its protein inhibitor (UGI), we combined rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). Computational docking of the unbound protein structures provides a list of possible three-dimensional models of the complex; DXMS identifies solvent-protected protein residues. DXMS showed that unbound hUNG is compactly folded, but unbound UGI is loosely packed. An increased level of solvent protection of hUNG in the complex was localized to four regions on the same face. The decrease in the number of incorporated deuterons was quantitatively interpreted as the minimum number of main-chain hUNG amides buried in the protein-protein interface. The level of deuteration of complexed UGI decreased throughout the protein chain, indicating both tighter packing and direct solvent protection by hUNG. Three UGI regions showing the greatest decreases were best interpreted leniently, requiring just one main-chain amide from each in the interface. Applying the DXMS constraints as filters to a list of docked complexes gave the correct complex as the largest favorable energy cluster. Thus, identification of approximate protein interfaces was sufficient to distinguish the protein complex. Surprisingly, incorporating the DXMS data as added favorable potentials in the docking calculation was less effective in finding the correct complex. The filtering method has greater flexibility, with the capability to test each constraint and enforce simultaneous contact by multiple regions, but with the caveat that the list from the unbiased docking must include correct complexes.

Macrophages detect pathogen infection via the activation of their plasma membrane-bound Toll-like receptor proteins (TLRs). The heterotypic interaction between the Toll/interleukin-1 receptor (TIR) domains of TLRs and adaptor proteins, likeMyeloid differentiation primary response gene 88 (MyD88), is the first intracellular step in the signaling pathway of the mammalian innate immune response. The hetero-oligomerization of the TIRs of the receptor and adaptor brings about the activation of the transcription factor NF-B, which regulates the synthesis of pro-inflammatory cytokines. Here, we report the first crystal structure of a bacterial TIR domain solved at 2.5 Å resolution. The three-dimensional fold of Paracoccus denitrificans TIR is identical to that observed for the TIR of human TLRs and MyD88 proteins. The structure shows a unique dimerization interface involving the DD-loop and EE-loop residues, whereas leaving the BB-loop highly exposed. Peptide amide hydrogendeuterium exchange mass spectrometry also reveals that the same region is used for dimerization in solution and in the context of the full-length protein. These results, together with a functional interaction between P. denitrificans TIR and MyD88 visualized in a co-immunoprecipitation assay, further substantiate the model that bacterial TIR proteins adopt structural mimicry of the host active receptor TIR domains to interfere with the signaling of TLRs and their adaptors to decrease the inflammatory response. Toll/interleukin-1 receptor (TIR)2 domain is a key mediator in the Toll-like receptor (TLR) signaling. TLRs are involved in early detection of pathogen-associated molecular patterns to enable quick responses to infection by triggering innate immune reactions through activation of the gene program regulated by the transcription factor nuclear factor-B (NF-B) and the recruitment of macrophages to the infection sites (1). The signaling of TLRs requires the homo-or heterodimerization of their extracellular leucine-rich repeats region mediated by the microbial pathogen-associated molecular patterns, leading to the dimerization of the receptor cytoplasmic TIR domains (2). Only in this active conformation are the receptor TIR domains capable of a functionally productive interaction with TIR domains of adaptor molecules, such as Myeloid differentiation primary response gene 88 (MyD88), MyD88 adaptor-like (Mal, also known as Toll/IL-1 receptor domain-containing adaptor protein (TIRAP)), TIR domain-containing adapter-inducing interferon-␤ (TRIF), or TRIF-related adaptor molecule (TRAM), to initiate the signaling cascade (3).Structures of TIR domains from human TLR1(4), TLR2(5), TLR10(6), IL-1RAPL(7), and MyD88 (Protein Data Bank (PDB) IDs: 2JS7; 2Z5V) have been determined, and they all showed a flavodoxin-like fold consisting of three layers ␣/␤/␣ with fivestranded parallel ␤-sheet ordered 2,1,3,4,5 surrounded by ␣-helices on each side. However, neither the homotypic interactions of TIR domains nor the heterotypic ones between the TIRs of receptors and a...

X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein–DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG–DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210–220 and 251–264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG–DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.