Biomedical subjects

Max H Nanao

Publications and source records attributed to Max H Nanao.

5 recordsLinked to original sources

PubMed2005

Improving radiation-damage substructures for RIP.

Specific radiation damage can be used to solve macromolecular structures using the radiation-damage-induced phasing (RIP) method. The method has been investigated for six disulfide-containing test structures (elastase, insulin, lysozyme, ribonuclease A, trypsin and thaumatin) using data sets that were collected on a third-generation synchrotron undulator beamline with a highly attenuated beam. Each crystal was exposed to the unattenuated X-ray beam between the collection of a 'before' and an 'after' data set. The X-ray 'burn'-induced intensity differences ranged from 5 to 15%, depending on the protein investigated. X-ray-susceptible substructures were determined using the integrated direct and Patterson methods in SHELXD. The best substructures were found by downscaling the 'after' data set in SHELXC by a scale factor K, with optimal values ranging from 0.96 to 0.99. The initial substructures were improved through iteration with SHELXE by the addition of negatively occupied sites as well as a large number of relatively weak sites. The final substructures ranged from 40 to more than 300 sites, with strongest peaks as high as 57sigma. All structures except one could be solved: it was not possible to find the initial substructure for ribonuclease A, however, SHELXE iteration starting with the known five most susceptible sites gave excellent maps. Downscaling proved to be necessary for the solution of elastase, lysozyme and thaumatin and reduced the number of SHELXE iterations in the other cases. The combination of downscaling and substructure iteration provides important benefits for the phasing of macromolecular structures using radiation damage.

Animals↗

PubMed2005

Phasing in the presence of radiation damage.

In the accurate estimation of small signals, redundancy of observations is often seen as an essential tool for the experimenter. This is particularly true during macromolecular structure determination by single-wavelength anomalous dispersion (SAD), where the exploitable signal can be less than a few percent. At the most intense undulator synchrotron beamlines, the effect of radiation damage can be such that all usable signal is obscured. Here the magnitude of this effect in experiments performed at the Se K-edge is quantified. Six successive data sets were collected on the same crystal, interspersed with two exposures to the X-ray beam during which data were not collected. It is shown that the very first data set has excellent phasing statistics, whereas these statistics degrade for the later data sets. Merging several data sets into one, highly redundant, data set only gave moderate improvements as a result of the presence of radiation damage. Part of the damage could be corrected for using a linear interpolation scheme. Interpolation of the data to a low-dose as well as to a high-dose data set allowed us to combine the SAD method with the radiation-damage induced phasing (RIP) technique, which further improved the experimental phases, especially after density modification. Some recommendations are given on how to mitigate the effect of radiation damage during structure determination.

Bacterial Proteins↗

PubMed2005

Structure of the kainate receptor subunit GluR6 agonist-binding domain complexed with domoic acid.

We report the crystal structure of the glycosylated ligand-binding (S1S2) domain of the kainate receptor subunit GluR6, in complex with the agonist domoate. The structure shows the expected overall homology with AMPA and NMDA receptor subunit structures but reveals an unexpected binding mode for the side chain of domoate, in which contact is made to the larger lobe only (lobe I). In common with the AMPA receptor subunit GluR2, the GluR6 S1S2 domain associates as a dimer, with many of the interdimer contacts being conserved. Subtle differences in these contacts provide a structural explanation for why GluR2 L483Y and GluR3 L507Y are nondesensitizing, but GluR6, which has a tyrosine at that site, is not. The structure incorporates native glycosylation, which has not previously been described for ionotropic glutamate receptors. The position of the sugars near the subunit interface rules out their direct involvement in subunit association but leaves open the possibility of indirect modulation. Finally, we observed several tetrameric assemblies that satisfy topological constraints with respect to connection to the receptor pore, and which are therefore candidates for the native quaternary structure.

Amino Acid Sequence↗

PubMed2004

Crystal structure of human otubain 2.

Ubiquitylation, the modification of cellular proteins by the covalent attachment of ubiquitin, is critical for diverse biological processes including cell cycle progression, signal transduction and stress response. This process can be reversed and regulated by a group of proteases called deubiquitylating enzymes (DUBs). Otubains are a recently identified family of DUBs that belong to the ovarian tumour (OTU) superfamily of proteins. Here, we report the first crystal structure of an OTU superfamily protein, otubain 2, at 2.1 A resolution and propose a model for otubain-ubiquitin binding on the basis of other DUB structures. Although otubain 2 is a member of the cysteine protease superfamily of folds, its crystal structure shows a novel fold for DUBs. Moreover, the active-site cleft is sterically occluded by a novel loop conformation resulting in an oxyanion hole, which consists uniquely of backbone amides, rather than the composite backbone/side-chain substructures seen in other DUBs and cysteine proteases. Furthermore, the residues that orient and stabilize the active-site histidine of otubain 2 are different from other cysteine proteases. This reorganization of the active-site topology provides a possible explanation for the low turnover and substrate specificity of the otubains.

Amino Acid Sequence↗

PubMed2003

Determining the basis of channel-tetramerization specificity by x-ray crystallography and a sequence-comparison algorithm: Family Values (FamVal).

We have developed a semiempirical algorithm called Family Values (FamVal), which identifies residues that encode functional specificity in a protein sequence. Given a multiple sequence alignment (MSA) grouped into functionally distinct subfamilies, FamVal calculates a specificity score for each subfamily at every amino acid position of an MSA. This algorithm was used to predict specificity-encoding positions within the tetramerization assembly (T1) domain of voltage-gated potassium (Kv) channel subfamilies Kv3 and Kv4. The importance of one such position (Arg to Ala at MSA position 93) was confirmed by in vitro pull-down assays. The structural basis of this assembly discrimination was elucidated by determining the crystal structure of the Kv4 T1 domain and comparing it to the Kv3 T1 domain.

Algorithms↗