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Lynn F Ten Eyck

Publications and source records attributed to Lynn F Ten Eyck.

13 recordsLinked to original sources
PubMed2006

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism.

The surface comparison of different serine-threonine and tyrosine kinases reveals a set of 30 residues whose spatial positions are highly conserved. The comparison between active and inactive conformations identified the residues whose positions are the most sensitive to activation. Based on these results, we propose a model of protein kinase activation. This model explains how the presence of a phosphate group in the activation loop determines the position of the catalytically important aspartate in the Asp-Phe-Gly motif. According to the model, the most important feature of the activation is a "spine" formation that is dynamically assembled in all active kinases. The spine is comprised of four hydrophobic residues that we detected in a set of 23 eukaryotic and prokaryotic kinases. It spans the molecule and plays a coordinating role in activated kinases. The spine is disordered in the inactive kinases and can explain how stabilization of the whole molecule is achieved upon phosphorylation.

Algorithms↗
PubMed2006

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis.

Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward- and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in EX1 kinetic events.

Amides↗
PubMed2006

Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments.

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high-resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side-chain hydrogens (present in MALDI-TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side-chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.

Amides↗
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

The cAMP binding domain: an ancient signaling module.

cAMP-binding domains from several different proteins were analyzed to determine the properties and interactions of this recognition motif. Systematic computational analyses, including structure-based sequence comparison, surface matching, affinity grid analysis, and analyses of the ligand protein interactions were carried out. These analyses show distinctive roles of the sugar phosphate and the adenine in the cAMP-binding module. We propose that the cAMP-binding regulatory proteins function by providing an allosteric system in which the presence or absence of cAMP produces a substantial structural change through the loss of hydrophobic interactions with the adenine ring and consequent repositioning of the C helix. The modified positioning of the helix in turn is recognized by a protein-binding event, completing the allostery.

Amino Acid Sequence↗
PubMed2004

Crystal structures of RIalpha subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3',5'-cyclic monophosphothioate and (Sp)-adenosine 3',5'-cyclic monophosphothioate, the phosphothioate analogues of cAMP.

Cyclic adenosine 5'-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R(p))-adenosine 3',5'-cyclic monophosphothioate ((R(p))-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S(p))-adenosine 3',5'-cyclic monophosphothioate ((S(p))-cAMPS) functions as an agonist. The crystal structures of a Delta(1-91) deletion mutant of the RIalpha regulatory subunit of PKA bound to (R(p))-cAMPS and (S(p))-cAMPS were determined at 2.4 and 2.3 A resolution, respectively. While the structures are similar to each other and to the crystal structure of RIalpha bound to cAMP, differences in the dynamical properties of the protein when (R(p))-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of Delta(1-91) RIalpha complexed with (R(p))-cAMPS phosphothioate analogue ((R(p))-RIalpha) compared to the cAMP- and (S(p))-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIalpha subunit can also be traced. This strand forms an intermolecular antiparallel beta-sheet with the same strand in an adjacent molecule and implies that the RIalpha subunit can form a weak homodimer even in the absence of its dimerization domain.

Binding Sites↗
PubMed2004

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.

Adenosine Triphosphatases↗
PubMed2003

Identification of the protein kinase A regulatory RIalpha-catalytic subunit interface by amide H/2H exchange and protein docking.

An important goal after structural genomics is to build up the structures of higher-order protein-protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and RIalpha regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen/deuterium (H/2H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A- and B-helices of RIalpha is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.

Amino Acid Sequence↗
PubMed2003

Finding needles in haystacks: Reranking DOT results by using shape complementarity, cluster analysis, and biological information.

We present an evaluation of our results for the first Critical Assessment of PRedicted Interaction (CAPRI). The methods used include the molecular docking program DOT, shape analysis tool FADE, cluster analysis and filtering based on biological data. Good results were obtained for most of the seven CAPRI targets, and for two systems, submissions having the highest number of correctly predicted contacts were produced.

Algorithms↗
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↗
PubMed2003

Protein structural alignment for detection of maximally conserved regions.

An algorithm for comparison of homologous protein structures and for study of conformational changes in proteins, has been developed. The method is based on identification of pieces of the two molecules that have similar shapes, as determined by the local conformation of the polypeptide chain. Pieces that superpose within a specified tolerance are assembled into domains based on similar transformations for superposition. The result is sets of pieces that represent conserved structural elements and conserved spatial relationships between structural elements within the proteins being compared. A similarity criterion based on maximum distance rather than on root mean square deviation reduces bias by outliers. The utility of the method is demonstrated by using examples from the protein kinase family.

Algorithms↗
PubMed2002

Hidden Markov models-based system (HMMSPECTR) for detecting structural homologies on the basis of sequential information.

HMMSPECTR is a tool for finding putative structural homologs for proteins with known primary sequences. HMMSPECTR contains four major components: a data warehouse with the hidden Markov models (HMM) and alignment libraries; a search program which compares the initial protein sequences with the libraries of HMMs; a secondary structure prediction and comparison program; and a dominant protein selection program that prepares the set of 10-15 "best" proteins from the chosen HMMs. The data warehouse contains four libraries of HMMs. The first two libraries were constructed using different HHM preparation options of the HAMMER program. The third library contains parts ("partial HMM") of initial alignments. The fourth library contains trained HMMs. We tested our program against all of the protein targets proposed in the CASP4 competition. The data warehouse included libraries of structural alignments and HMMs constructed on the basis of proteins publicly available in the Protein Data Bank before the CASP4 meeting. The newest fully automated versions of HMMSPECTR 1.02 and 1.02ss produced better results than the best result reported at CASP4 either by r.m.s.d. or by length (or both) in 64% (HMMSPECTR 1.02) and 79% (HMMSPECTR 1.02ss) of the cases. The improvement is most notable for the targets with complexity 4 (difficult fold recognition cases).

Markov Chains↗