Albert Y. Lau

Crystal Structure of a Human Alkylbase-DNA Repair Enzyme Complexed to DNA: Mechanisms for Nucleotide Flipping and Base Excision

DNA N-glycosylases are base excision-repair proteins that locate and cleave damaged bases from DNA as the first step in restoring the genetic blueprint. The human enzyme 3-methyladenine DNA glycosylase removes a diverse group of damaged bases from DNA, including cytotoxic and mutagenic alkylation adducts of purines. We report the crystal structure of human 3-methyladenine DNA glycosylase complexed to a mechanism-based pyrrolidine inhibitor. The enzyme has intercalated into the minor groove of DNA, causing the abasic pyrrolidine nucleotide to flip into the enzyme active site, where a bound water is poised for nucleophilic attack. The structure shows an elegant means of exposing a nucleotide for base excision as well as a network of residues that could catalyze the in-line displacement of a damaged base from the phosphodeoxyribose backbone.

3-Methyladenine DNA glycosylases: structure, function, and biological importance

The genome continuously suffers damage due to its reactivity with chemical and physical agents. Finding such damage in genomes (that can be several million to several billion nucleotide base pairs in size) is a seemingly daunting task. 3‐Methyladenine DNA glycosylases can initiate the base excision repair (BER) of an extraordinarily wide range of substrate bases. The advantage of such broad substrate recognition is that these enzymes provide resistance to a wide variety of DNA damaging agents; however, under certain circumstances, the eclectic nature of these enzymes can confer some biological disadvantages. Solving the X‐ray crystal structures of two 3‐methyladenine DNA glycosylases, and creating cells and animals altered for this activity, contributes to our understanding of their enzyme mechanism and how such enzymes influence the biological response of organisms to several different types of DNA damage. BioEssays 21:668–676, 1999.

Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations

Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein, highlighting a crucial role for the C-terminal, BRCT repeat region in mediating BRCA1 tumor suppressor function. However, the molecular and genetic effects of missense mutations that map to the BRCT region remain largely unknown. Using a protease-based assay, we directly assessed the sensitivity of the folding of the BRCT domain to an extensive set of truncation and single amino acid substitutions derived from breast cancer screening programs. The protein can tolerate truncations of up to 8 amino acids, but further deletion results in drastic BRCT folding defects. This molecular phenotype can be correlated with an increased susceptibility to disease. A cross-validated computational assessment of the BRCT mutation data base suggests that as much as half of all BRCT missense mutations contribute to BRCA1 loss of function and disease through protein-destabilizing effects. The coupled use of proteolytic methods and computational predictive methods to detect mutant BRCA1 conformations at the protein level will augment the efficacy of current BRCA1 screening protocols, especially in the absence of clinical data that can be used to discriminate deleterious BRCT missense mutations from benign polymorphisms.

The Hidden Energetics of Ligand-Binding and Activation in a Glutamate Receptor

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate most excitatory synaptic transmission in the central nervous system. The free energy of neurotransmitter binding to the ligand-binding domains (LBDs) of iGluRs is converted into useful work to drive receptor activation. We have computed the principal thermodynamic contributions from ligand docking and ligand-induced closure of LBDs for nine ligands of GluA2 using all-atom molecular dynamics free energy simulations. We have validated the results by comparison with experimentally measured apparent affinities to the isolated LBD. Features in the free energy landscapes that govern closure of LBDs are key determinants of binding free energies. An analysis of accessible LBD conformations transposed into the context of an intact GluA2 receptor revealed that the relative displacement of specific diagonal subunits in the tetrameric structure may be key to the action of partial agonists.

Nanosculpting reversed wavelength sensitivity into a photoswitchable iGluR

Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine the efficacy of PTL gating in the ionotropic glutamate receptor iGluR6 using a family of photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs that covalently attach to the clamshell ligand-binding domain. Experiments and molecular dynamics simulations of the modified proteins show that optical switching depends on 2 factors: (i) the relative occupancy of the binding pocket in the 2 photoisomers of MAG and (ii) the degree of clamshell closure that is possible given the disposition of the MAG linker. A synthesized short version of MAG turns the channel on in either the cis or trans state, depending on the point of attachment. This yin/yang optical control makes it possible for 1 wavelength of light to elicit action potentials in one set of neurons, while deexciting a second set of neurons in the same preparation, whereas a second wavelength has the opposite effect. The ability to generate opposite responses with a single PTL and 2 versions of a target channel, which can be expressed in different cell types, paves the way for engineering opponency in neurons that mediate opposing functions.

Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase.

Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.

Conformational Analysis of NMDA Receptor GluN1, GluN2, and GluN3 Ligand-Binding Domains Reveals Subtype-Specific Characteristics

The NMDA receptor family of glutamate receptor ion channels is formed by obligate heteromeric assemblies of GluN1, GluN2, and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states unexpectedly reveal open- and closed-cleft conformations, respectively, with water molecules filling the binding pockets. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo-state LBDs sample closed-cleft conformations, suggesting that their agonists bind via a conformational selection mechanism. By contrast, free energy landscapes for the AMPA receptor GluA2 LBD suggest binding of glutamate via an induced-fit mechanism. Principal component analysis reveals a rich spectrum of hinge bending, rocking, twisting, and sweeping motions that are different for the GluN1, GluN2A, GluN3A, and GluA2 LBDs. This variation highlights the structural complexity of signaling by glutamate receptor ion channels.

A structural model for K2P potassium channels based on 23 pairs of interacting sites and continuum electrostatics.

K2PØ, the two-pore domain potassium background channel that determines cardiac rhythm in Drosophila melanogaster, and its homologues that establish excitable membrane activity in mammals are of unknown structure. K2P subunits have two pore domains flanked by transmembrane (TM) spans: TM1-P1-TM2-TM3-P2-TM4. To establish spatial relationships in K2PØ, we identified pairs of sites that display electrostatic compensation. Channels silenced by the addition of a charge in pore loop 1 (P1) or P2 were restored to function by countercharges at specific second sites. A three-dimensional homology model was determined using the crystal structure of KV1.2, effects of K2PØ mutations to establish alignment, and compensatory charge–charge pairs. The model was refined and validated by continuum electrostatic free energy calculations and covalent linkage of introduced cysteines. K2P channels use two subunits arranged so that the P1 and P2 loops contribute to one pore, identical P loops face each other diagonally across the pore, and the channel complex has bilateral symmetry with a fourfold symmetric selectivity filter.

A Conformational Intermediate in Glutamate Receptor Activation

Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an ∼30° rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

Crystallizing thoughts about DNA base excision repair

Chemically damaged bases are removed from DNA by glycosylases that locate the damage and cleave the bond between the modified base and the deoxyribose sugar of the DNA backbone. The detection of damaged bases in DNA poses two problems: (1) The aberrant bases are mostly buried within the double helix, and (2) a wide variety of chemically different modifications must be efficiently recognized and removed. The human alkyladenine glycosylase (AAG) and Escherichia coli Alka DNA glycosylases excise many different types of alkylated bases from DNA. Crystal structures of these enzymes show how substrate bases are exposed to the enzyme active site and they suggest mechanisms of catalytic specificity. Both enzymes bend DNA and flip substrate bases out of the double helix and into the enzyme active site for cleavage. Although AAG and AlkA have very different overall folds, some common features of their substrate-binding sites suggest related strategies for the selective recognition of a chemically diverse group of alkylated substrates.