Biomedical subjects

Nguyen-Huu Xuong

Publications and source records attributed to Nguyen-Huu Xuong.

7 recordsLinked to original sources

PubMed2006

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Active pixel sensor array as a detector for electron microscopy.

A new high-resolution recording device for transmission electron microscopy (TEM) is urgently needed. Neither film nor CCD cameras are systems that allow for efficient 3-D high-resolution particle reconstruction. We tested an active pixel sensor (APS) array as a replacement device at 200, 300, and 400 keV using a JEOL JEM-2000 FX II and a JEM-4000 EX electron microscope. For this experiment, we used an APS prototype with an area of 64 x 64 pixels of 20 microm x 20 microm pixel pitch. Single-electron events were measured by using very low beam intensity. The histogram of the incident electron energy deposited in the sensor shows a Landau distribution at low energies, as well as unexpected events at higher absorbed energies. After careful study, we concluded that backscattering in the silicon substrate and re-entering the sensitive epitaxial layer a second time with much lower speed caused the unexpected events. Exhaustive simulation experiments confirmed the existence of these back-scattered electrons. For the APS to be usable, the back-scattered electron events must be eliminated, perhaps by thinning the substrate to less than 30 microm. By using experimental data taken with an APS chip with a standard silicon substrate (300 microm) and adjusting the results to take into account the effect of a thinned silicon substrate (30 microm), we found an estimate of the signal-to-noise ratio for a back-thinned detector in the energy range of 200-400 keV was about 10:1 and an estimate for the spatial resolution was about 10 microm.

Cryoelectron Microscopy↗

PubMed2005

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.

Binding Sites↗

PubMed2005

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.

Adenosine Triphosphate↗

PubMed2004

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.

Allosteric Site↗

PubMed2003

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.

Adenosine↗

PubMed2002

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.

Adenosine Diphosphate↗