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1.  The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution* 
The Journal of Biological Chemistry  2012;287(39):32381-32393.
Background: NDE1 and NDEL1 are neurodevelopmental and mitotic proteins with extended coiled-coil N termini, but unknown C-terminal structure.
Results: Recombinant NDE1/NDEL1 form dimers and tetramers in which their C termini interact with their N-terminal domains.
Conclusion: NDE1/NDEL1 each adopt a sharply bent back structure.
Significance: This explains the existence of two distinct dynein-binding domains on NDE1/NDEL1 and instability of disease-associated mutants lacking C termini.
Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.
PMCID: PMC3463352  PMID: 22843697
Electron Microscopy (EM); Homology Modeling; Mass Spectrometry (MS); Neurodevelopment; Protein Cross-linking; Protein Folding; Protein Structure; NDE1; NDEL1
2.  The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein 
Nucleic Acids Research  2008;37(3):762-770.
Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.
PMCID: PMC2647291  PMID: 19074193

Results 1-2 (2)