Sandy Harper

The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate

Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression. The structure of an ESA1–CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins. Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate. This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily. However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone. These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors.

Cooperativity in Forced Unfolding of Tandem Spectrin Repeats

Force-driven conformational changes provide a broad basis for protein extensibility, and multidomain proteins broaden the possibilities further by allowing for a multiplicity of forcibly extended states. Red cell spectrin is prototypical in being an extensible, multidomain protein widely recognized for its contribution to erythrocyte flexibility. Atomic force microscopy has already shown that single repeats of various spectrin family proteins can be forced to unfold reversibly under extension. Recent structural data indicates, however, that the linker between triple-helical spectrin repeats is often a contiguous helix, thus raising questions as to what the linker contributes and what defines a domain mechanically. We have examined the extensible unfolding of red cell spectrins as monomeric constructs of just two, three, or four repeats from the actin-binding ends of both alpha- and beta-chains, i.e., alpha(18-21) and beta(1-4) or their subfragments. In addition to single repeat unfolding evident in sawtooth patterns peaked at relatively low forces (<50 pN at 1 nm/ms extension rates), tandem repeat unfolding is also demonstrated in ensemble-scale analyses of thousands of atomic force microscopy contacts. Evidence for extending two chains and loops is provided by force versus length scatterplots which also indicate that tandem repeat unfolding occurs at a significant frequency relative to single repeat unfolding. Cooperativity in forced unfolding of spectrin is also clearly demonstrated by a common force scale for the unfolding of both single and tandem repeats.

Pathway shifts and thermal softening in temperature-coupled forced unfolding of spectrin domains.

Pathways of unfolding a protein depend in principle on the perturbation-whether it is temperature, denaturant, or even forced extension. Widely-shared, helical-bundle spectrin repeats are known to melt at temperatures as low as 40-45 degrees C and are also known to unfold via multiple pathways as single molecules in atomic force microscopy. Given the varied roles of spectrin family proteins in cell deformability, we sought to determine the coupled effects of temperature on forced unfolding. Bimodal distributions of unfolding intervals are seen at all temperatures for the four-repeat beta(1-4) spectrin-an alpha-actinin homolog. The major unfolding length corresponds to unfolding of a single repeat, and a minor peak at twice the length corresponds to tandem repeats. Increasing temperature shows fewer tandem events but has no effect on unfolding intervals. As T approaches T(m), however, mean unfolding forces in atomic force microscopy also decrease; and circular dichroism studies demonstrate a nearly proportional decrease of helical content in solution. The results imply a thermal softening of a helical linker between repeats which otherwise propagates a helix-to-coil transition to adjacent repeats. In sum, structural changes with temperature correlate with both single-molecule unfolding forces and shifts in unfolding pathways.

Cdc13 N-Terminal Dimerization, DNA Binding, and Telomere Length Regulation

ABSTRACT The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

Cdc13 OB2 dimerization required for productive Stn1 binding and efficient telomere maintenance.

Cdc13 is an essential yeast protein required for telomere length regulation and genome stability. It does so via its telomere-capping properties and by regulating telomerase access to the telomeres. The crystal structure of the Saccharomyces cerevisiae Cdc13 domain located between the recruitment and DNA binding domains reveals an oligonucleotide-oligosaccharide binding fold (OB2) with unusually long loops extending from the core of the protein. These loops are involved in extensive interactions between two Cdc13 OB2 folds leading to stable homodimerization. Interestingly, the functionally impaired cdc13-1 mutation inhibits OB2 dimerization. Biochemical assays indicate OB2 is not involved in telomeric DNA or Stn1 binding. However, disruption of the OB2 dimer in full-length Cdc13 affects Cdc13-Stn1 association, leading to telomere length deregulation, increased temperature sensitivity, and Stn1 binding defects. We therefore propose that dimerization of the OB2 domain of Cdc13 is required for proper Cdc13, Stn1, Ten1 (CST) assembly and productive telomere capping.