Jason W. O'Neill

Mcl-1 is required for Akata6 B-lymphoma cell survival and is converted to a cell death molecule by efficient caspase-mediated cleavage

Enforced expression of the antiapoptotic Bcl-2 family protein Mcl-1 promotes lymphomagenesis in the mouse; however, the functional role of Mcl-1 in human B-cell lymphoma remains unclear. We demonstrate that Mcl-1 is widely expressed in malignant B-cells, and high-level expression of Mcl-1 is required for B-lymphoma cell survival, since transfection of Mcl-1-specific antisense oligodeoxynucleotides was sufficient to promote apoptosis in Akata6 lymphoma cells. Mcl-1 was efficiently cleaved by caspases at evolutionarily conserved aspartic acid residues in vitro, and during cisplatin-induced apoptosis in B-lymphoma cell lines and spontaneous apoptosis of primary malignant B-cells. Overexpression of the Mcl-1 cleavage product that accumulated during apoptosis was sufficient to kill cells. Therefore, Mcl-1 is an essential survival molecule for B-lymphoma cells and is cleaved by caspases to a death-promoting molecule during apoptosis. In contrast to Mcl-1, Bcl-2 and Bcl-XL were relatively resistant to caspase cleavage in vitro and in intact cells. Interfering with Mcl-1 function appears to be an effective means of inducing apoptosis in Mcl-1-positive B-cell lymphoma, and the unique sensitivity of Mcl-1 to caspase-mediated cleavage suggests an attractive strategy for converting it to a proapoptotic molecule.

Crystal structure of E. coli β–carbonic anhydrase, an enzyme with an unusual pH–dependent activity

Carbonic anhydrases fall into three distinct evolutionary and structural classes: α, β, and γ. The β‐class carbonic anhydrases (β‐CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a β‐CA from Escherichia coli, to a resolution of 2.0 Å. In agreement with the structure of the β‐CA from the chloroplast of the red alga Porphyridium purpureum, the active‐site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some β‐CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH‐dependent in the physiological range, and its expression in yeast complements an oxygen‐sensitive phenotype displayed by a β‐CA‐deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other β‐CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.

Conversion of monomeric protein L to an obligate dimer by computational protein design

Protein L consists of a single α-helix packed on a four-stranded β-sheet formed by two symmetrically opposed β-hairpins. We use a computer-based protein design procedure to stabilize a domain-swapped dimer of protein L in which the second β-turn straightens and the C-terminal strand inserts into the β-sheet of the partner. The designed obligate dimer contains three mutations (A52V, N53P, and G55A) and has a dissociation constant of ≈700 pM, which is comparable to the dissociation constant of many naturally occurring protein dimers. The structure of the dimer has been determined by x-ray crystallography and is close to the in silico model.

Bcl-XL mutations suppress cellular sensitivity to antimycin A.

Cells expressing high levels of the BCL-XL anti-apoptotic protein are preferentially killed by the mitochondrial inhibitor antimycin A (AA). Computational modeling predicts a binding site for AA in the extended hydrophobic groove on BCL-XL, previously identified as an interface for dimerization to BAX and related proapoptotic proteins. Here, we identify BCL-XL hydrophobic groove mutants with normal cellular anti-apoptotic function but suppressed sensitivity to AA. The LD50 of AA for cells expressing BCL-XL mutants directly correlates with the measured in vitro dissociation constants for AA binding. These results indicate that BCL-XL is a principal target mediating AA cytotoxicity.

Structures of the B1 domain of protein L from Peptostreptococcus magnus with a tyrosine to tryptophan substitution

The three-dimensional structure of a tryptophan-containing variant of the IgG-binding B1 domain of protein L has been solved in two crystal forms to 1.7 and 1.8 A resolution. In one of the crystal forms, the entire N-terminal histidine-tag region was immobilized through the coordination of zinc ions and its structural conformation along with the zinc coordination scheme were determined. However, the ordering of the histidine tag by zinc does not affect the overall structure of the rest of the protein. Structural comparisons of the tryptophan-containing variant with an NMR-derived wild-type structure, which contains a tyrosine at position 47, reveals a common fold, although the overall backbone root-mean-square difference is 1.5 A. The Y47W substitution only caused local rearrangement of several side chains, the most prominent of which is the rotation of the Tyr34 side chain, resulting in a 6 A displacement of its hydroxyl group. A small methyl-sized cavity bounded by beta-strands 1, 2 and 4 and the alpha-helix was found in the structures of the Y47W-substituted protein L B1 domain. This cavity may be created as the result of subsequent side-chain rearrangements caused by the Y47W substitution. These high-resolution structures of the tryptophan-containing variant provide a reference frame for the analysis of thermodynamic and kinetic data derived from a series of mutational studies of the protein L B1 domain.

Single-site mutations induce 3D domain swapping in the B1 domain of protein L from Peptostreptococcus magnus.

BACKGROUND Thermodynamic and kinetic studies of the Protein L B1 domain (Ppl) suggest a folding pathway in which, during the folding transition, the first beta hairpin is formed while the second beta hairpin and the alpha helix are largely unstructured. The same mutations in the two beta turns have opposite effects on the folding and unfolding rates. Three of the four residues composing the second beta turn in Ppl have consecutive positive phi angles, indicating strain in the second beta turn. RESULTS We have determined the crystal structures of the beta turn mutants G55A, K54G, and G15A, as well as a core mutant, V49A, in order to investigate how backbone strain affects the overall structure of Ppl. Perturbation of the hydrophobic interactions at the closed interface by the V49A mutation triggered the domain swapping of the C-terminal beta strand that relieved the strain in the second beta turn. Interestingly, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escalated the strain in the second beta turn, and this increased strain shifted the equilibrium toward the domain-swapped dimer. The K54G structure revealed that the increased stability is due to the reduction of strain in the second beta turn, while the G15A structure showed that increased strain alone is insufficient to trigger domain swapping. CONCLUSIONS Domain swapping in Ppl is determined by the balance of two opposing components of the free energy. One is the strain in the second beta turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A single-site mutation can disrupt this balance and trigger domain swapping.

Cloning, crystallization and preliminary characterization of a β-carbonic anhydrase from Escherichia coli

Carbonic anhydrases are zinc metalloenzymes that fall into three distinct evolutionary and structural classes, alpha, beta and gamma. Although alpha-class enzymes, particularly mammalian carbonic anhydrase II, have been the subject of extensive structural studies, for the beta class, consisting of a wide variety of prokaryotic and plant chloroplast carbonic anhydrases, the structural data is quite limited. A member of the beta class from E. coli (CynT2) has been crystallized in native and selenomethionine-labelled forms and multiwavelength anomalous dispersion techniques have been applied in order to determine the positions of anomalous scatterers. The resulting phase information is sufficient to produce an interpretable electron-density map. A crystal structure for CynT2 would contribute significantly to the emerging structural knowledge of a biologically important class of enzymes that perform critical functions in carbon fixation and prokaryotic metabolism.

Bcl-2-related proteins as drug targets.

The Bcl-2 family of proteins provide the most unambiguous link between mitochondrial functions and apoptosis, as their only (or principal) functions appear to be as regulators of this cell death pathway. Rational drug design to manipulate the functions of these proteins has been hampered by the lack of a clear understanding of a biochemical or molecular function, with disruption of intra-family protein-protein interactions as the only known, but daunting, objective. There has been substantial progress in this task using molecular modeling and drug leads. The prospects are also good for development of chemical tools for functional analysis of the Bcl-2 proteins.

Crystallization and preliminary X-ray diffraction studies of mutants of B1 IgG-binding domain of protein L from Peptostreptococcus magnus.

The small 62-residue IgG-binding domain B1 of protein L from Peptostreptococcus magnus (Ppl-B1) has proven to be a simple system for the study of the thermodynamics and kinetics of protein folding. X-ray diffraction studies have been initiated in order to determine how the thermostability, folding and unfolding rates of a series of point mutations spanning Ppl-B1 correlate with the high-resolution structures. To this end, a tryptophan-containing variant of Ppl-B1 (herein known as wild type) and two mutants, Lys61Ala and Val49Ala, have been crystallized. Full data sets have been collected for the wild type and the Lys61Ala and Val49Ala mutants to resolutions of 1. 7, 2.3 and 1.8 A, respectively. Interestingly, all three crystallize using different precipitants and in different space groups. This may be a consequence of the relatively large effects of single-site mutations on surface-charge distribution or structural conformation, which might affect crystal contact sites.