Improving signal to noise in labeled biological specimens using energy-filtered TEM of sections with a drift correction strategy and a direct detection device.

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD's) to increase the signal to noise as compared with CCD's. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.

A dynamic mechanism for AKAP binding to RII isoforms of cAMP-dependent protein kinase.

A kinase-anchoring proteins (AKAPs) target PKA to specific microdomains by using an amphipathic helix that docks to N-terminal dimerization and docking (D/D) domains of PKA regulatory (R) subunits. To understand specificity, we solved the crystal structure of the helical motif from D-AKAP2, a dual-specific AKAP, bound to the RIIalpha D/D domain. The 1.6 Angstrom structure reveals how this dynamic, hydrophobic docking site is assembled. A stable, hydrophobic docking groove is formed by the helical interface of two RIIalpha protomers. The flexible N terminus of one protomer is then recruited to the site, anchored to the peptide through two essential isoleucines. The other N terminus is disordered. This asymmetry provides greater possibilities for AKAP docking. Although there is strong discrimination against RIalpha in the N terminus of the AKAP helix, the hydrophobic groove discriminates against RIIalpha. RIalpha, with a cavity in the groove, can accept a bulky tryptophan, whereas RIIalpha requires valine.

Crystal structure of the E230Q mutant of cAMP-dependent protein kinase reveals an unexpected apoenzyme conformation and an extended N-terminal A helix.

Glu230, one of the acidic residues that cluster around the active site of the catalytic subunit of cAMP-dependent protein kinase, plays an important role in substrate recognition. Specifically, its side chain forms a direct salt-bridge interaction with the substrate's P-2 Arg. Previous studies showed that mutation of Glu230 to Gln (E230Q) caused significant decreases not only in substrate binding but also in the rate of phosphoryl transfer. To better understand the importance of Glu230 for structure and function, we solved the crystal structure of the E230Q mutant at 2.8 A resolution. Surprisingly, the mutant preferred an open conformation with no bound ligands observed, even though the crystals were grown in the presence of MgATP and the inhibitor peptide, IP20. This is in contrast to the wild-type protein that, under the same conditions, prefers the closed conformation of a ternary complex. The structure highlights the importance of the electrostatic surface not only for substrate binding and catalysis, but also for the mechanism for closing the active site cleft. This surface mutation clearly disrupts the recognition and binding of substrate peptide so that the enzyme prefers an open conformation that cannot trap ATP. This is consistent with the reinforcing concepts of conformational dynamics and the synergistic binding of ATP and substrate peptide. Another unusual feature of the structure is the observation of the entire N terminus (Gly1-Thr32) assumes an extended alpha-helix conformation. Finally, based on temperature factors, this mutant structure is more stable than the wild-type C-subunit in the apo state.

Crystal structure of a complex between the catalytic and regulatory (RIalpha) subunits of PKA.

The 2.0-angstrom structure of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) catalytic subunit bound to a deletion mutant of a regulatory subunit (RIalpha) defines a previously unidentified extended interface. The complex provides a molecular mechanism for inhibition of PKA and suggests how cAMP binding leads to activation. The interface defines the large lobe of the catalytic subunit as a stable scaffold where Tyr247 in the G helix and Trp196 in the phosphorylated activation loop serve as anchor points for binding RIalpha. These residues compete with cAMP for the phosphate binding cassette in RIalpha. In contrast to the catalytic subunit, RIalpha undergoes major conformational changes when the complex is compared with cAMP-bound RIalpha. The inhibitor sequence docks to the active site, whereas the linker, also disordered in free RIalpha, folds across the extended interface. The beta barrel of cAMP binding domain A, which is the docking site for cAMP, remains largely intact in the complex, whereas the helical subdomain undergoes major reorganization.

RIalpha subunit of PKA: a cAMP-free structure reveals a hydrophobic capping mechanism for docking cAMP into site B.

In eukaryotes the primary target for cAMP, a ubiquitous second messenger, is cAMP-dependent protein kinase (PKA). Understanding how binding and release of cAMP changes the cAMP binding domains and then triggers long-range allosteric responses is an important challenge. This conformational switching requires structure solutions of cAMP binding domains in cAMP-bound and cAMP-free states. We describe for the first time a crystal structure of the cAMP binding domains of PKA type Ialpha regulatory subunit where site A is occupied by cGMP and site B is unoccupied. The structure reveals that the carboxyl terminus of domain B serves as a hydrophobic cap, locking the cyclic nucleotide via its adenine ring into the beta-barrel. In the absence of cAMP, the "cap" is released via an extension of the C-terminal helix. This simple hinge mechanism for binding and release of cAMP also provides a mechanism for allosteric communication between sites A and B.

Crystal structure of a cAMP-dependent protein kinase mutant at 1.26A: new insights into the catalytic mechanism.

The catalytic subunit of cAMP-dependent protein kinase has served as a paradigm for the entire kinase family. In the course of studying the structure-function relationship of the P+1 loop (Leu198-Leu205) of the kinase, we have solved the crystal structure of the Tyr204 to Ala mutant in complexes with Mg.ATP and an inhibitory peptide at 1.26A, with overall structure very similar to that of the wild-type protein. However, at the nucleotide binding site, ATP was found largely hydrolyzed, with the products ADP-PO(4) retained in the structure. High-resolution refinement suggests that 26% of the molecules contain the intact ATP, whereas 74% have the hydrolyzed products. The observation of the substrate and product states in the same structure adds significant information to our understanding of the phosphoryl transfer process. Structural examination of the mutation site substantiates and extends the emerging concept that the hydrophobic core in the large lobe of the kinase might serve as a stable platform for anchoring key segments involved in catalysis. We propose that Tyr204 is critical for anchoring the P+1 loop to the core. Further analysis has highlighted two major connections between the P+1 loop and the catalytic loop (Arg165-Asn171). One emphasizes the hydrophobic packing of Tyr204 and Leu167 mediated through residues from the alphaF-helix, recently recognized as a signal integration motif, which together with the alphaE-helix forms the center of the hydrophobic core network. The other connection is mediated by the hydrogen bond interaction between Thr201 and Asp166, in a substrate-dependent manner. We speculate that the latter interaction may be important for the kinase to sense the presence of substrate and prepare itself for the catalytic reaction. Thus, the P+1 loop is not merely involved in substrate binding; it mediates the communication between substrate and catalytic residues.

Balanol analogues probe specificity determinants and the conformational malleability of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase catalytic subunit.

The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selective-characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3',5'-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium- and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.

Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure.

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.

Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase.

To understand the molecular mechanism underlying phosphoryl transfer of cAMP-dependent protein kinase, the structure of the catalytic subunit in complex with ADP, aluminum fluoride, Mg2+ ions and a substrate peptide was determined at 2.0 A resolution. Aluminum fluoride was modeled as AlF3 in a planar geometry; it is positioned 2.3 A from both the donor oxygen of ADP and the hydroxyl group of the recipient Ser residue. In this configuration, the aluminum atom forms a trigonal bipyramidal coordination with the oxygen atoms of the donor and recipient groups at the apical positions. This arrangement suggests that aluminum fluoride mimics the transition state and provides the first direct structural evidence for the in-line mechanism of phosphoryl transfer in a protein kinase.