Biomedical subjects

Elizabeth A Komives

Publications and source records attributed to Elizabeth A Komives.

At least 19 recordsLinked to original sources

PubMed2006

Regions of IkappaBalpha that are critical for its inhibition of NF-kappaB.DNA interaction fold upon binding to NF-kappaB.

Nuclear factor kappaB (NF-kappaB) transcription factors regulate genes responsible for critical cellular processes. IkappaBalpha, -beta, and -epsilon bind to NF-kappaBs and inhibit their transcriptional activity. The NF-kappaB-binding domains of IkappaBs contain six ankyrin repeats (ARs), which adopt a beta-hairpin/alpha-helix/loop/alpha-helix/loop architecture. IkappaBalpha appears compactly folded in the IkappaBalpha.NF-kappaB crystal structure, but biophysical studies suggested that IkappaBalpha might be flexible even when bound to NF-kappaB. Amide H/(2)H exchange in free IkappaBalpha suggests that ARs 2-4 are compact, but ARs 1, 5, and 6 are conformationally flexible. Amide H/(2)H exchange is one of few techniques able to experimentally identify regions that fold upon binding. Comparison of amide H/(2)H exchange in free and NF-kappaB-bound IkappaBalpha reveals that the beta-hairpins in ARs 5 and 6 fold upon binding to NF-kappaB, but AR 1 remains highly solvent accessible. These regions are implicated in various aspects of NF-kappaB regulation, such as controlling degradation of IkappaBalpha, enabling high-affinity interaction with different NF-kappaB dimers, and preventing NF-kappaB from binding to its target DNA. Thus, IkappaBalpha conformational flexibility and regions of IkappaBalpha folding upon binding to NF-kappaB are important attributes for its regulation of NF-kappaB transcriptional activity.

Amides↗

PubMed2006

Stabilizing IkappaBalpha by "consensus" design.

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

Amino Acid Sequence↗

PubMed2006

Solvent accessibility of protein surfaces by amide H/2H exchange MALDI-TOF mass spectrometry.

One advantage of detecting amide H/2H exchange by mass spectrometry instead of NMR is that the more rapidly exchanging surface amides are still detectable. In this study, we present quench-flow amide H/2H exchange experiments to probe how rapidly the surfaces of two different proteins exchange. We compared the amide H/2H exchange behavior of thrombin, a globular protein, and IkappaBalpha, a nonglobular protein, to explore any differences in the determinants of amide H/2H exchange rates for each class of protein. The rates of exchange of only a few of the surface amides were as rapid as the "intrinsic" exchange rates measured for amides in unstructured peptides. Most of the surface amides exchanged at a slower rate, despite the fact that they were not seen to be hydrogen bonded to another protein group in the crystal structure. To elucidate the influence of the surface environment on amide H/2H exchange, we compared exchange data with the number of amides participating in hydrogen bonds with other protein groups and with the solvent accessible surface area. The best correlation with amide H/2H exchange was found with the total solvent accessible surface area, including side chains. In the case of the globular protein, the correlation was modest, whereas it was well correlated for the nonglobular protein. The nonglobular protein also showed a correlation between amide exchange and hydrogen bonding. These data suggest that other factors, such as complex dynamic behavior and surface burial, may alter the expected exchange rates in globular proteins more than in nonglobular proteins where all of the residues are near the surface.

Amides↗

PubMed2006

Amide H/2H exchange reveals a mechanism of thrombin activation.

Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.

Amides↗

PubMed2006

Thermodynamics reveal that helix four in the NLS of NF-kappaB p65 anchors IkappaBalpha, forming a very stable complex.

IkappaBalpha is an ankyrin repeat protein that inhibits NF-kappaB transcriptional activity by sequestering NF-kappaB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IkappaBalpha ankyrin repeat domain to NF-kappaB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IkappaBalpha and NF-kappaB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K(D,obs) of approximately 40 pM at 37 degrees C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-kappaB(p50/p65) heterodimer and p65 homodimer bound IkappaBalpha with almost indistinguishable thermodynamics, except that the NF-kappaB p65 homodimer was characterized by a more favorable DeltaH(obs) relative to the NF-kappaB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (DeltaC(P,obs)), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

Animals↗

PubMed2006

An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis.

Phytochelatins (PCs) are peptides that function in heavy-metal chelation and detoxification in plants and fungi. A recent study showed that PCs have the ability to undergo long-distance transport in a root-to-shoot direction in transgenic Arabidopsis (Arabidopsis thaliana). To determine whether long-distance transport of PCs can occur in the opposite direction, from shoots to roots, the wheat (Triticum aestivum) PC synthase (TaPCS1) gene was expressed under the control of a shoot-specific promoter (CAB2) in an Arabidopsis PC-deficient mutant, cad1-3 (CAB2TaPCS1/cad1-3). Analyses demonstrated that TaPCS1 is expressed only in shoots and that CAB2TaPCS1/cad1-3 lines complement the cadmium (Cd) and arsenic metal sensitivity of cad1-3 shoots. CAB2TaPCS1/cad1-3 plants exhibited higher Cd accumulation in roots and lower Cd accumulation in shoots compared to wild type. Fluorescence HPLC coupled to mass spectrometry analyses directly detected PC2 in the roots of CAB2:TaPCS1/cad1-3 but not in cad1-3 controls, suggesting that PC2 is transported over long distances in the shoot-to-root direction. In addition, wild-type shoot tissues were grafted onto PC synthase cad1-3 atpcs2-1 double loss-of-function mutant root tissues. An Arabidopsis grafting technique for mature plants was modified to obtain an 84% success rate, significantly greater than a previous rate of approximately 11%. Fluorescence HPLC-mass spectrometry showed the presence of PC2, PC3, and PC4 in the root tissue of grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots, demonstrating that PCs are transported over long distances from shoots to roots in Arabidopsis.

Arabidopsis↗

PubMed2006

Ligand-induced conformational changes in the acetylcholine-binding protein analyzed by hydrogen-deuterium exchange mass spectrometry.

Recent x-ray crystallographic studies of the acetylcholine-binding protein (AChBP) suggest that loop C, found at the circumference of the pentameric molecule, shows distinctive conformational changes upon antagonist and agonist occupation. We have employed hydrogen-deuterium exchange mass spectrometry to examine the influence of bound ligands on solvent exposure of AChBP. Quantitative measurements of deuterium incorporation are possible for approximately 56% of the Lymnaea AChBP sequence, covering primarily the outer surface of AChBP. In the apoprotein, two regions flanking the ligand occupation site at the subunit interface, loop C (residues 175-193) and loop F (residues 164-171), show greater extents of solvent exchange than other regions of the protein including the N- and C-terminal regions. Occupation by nicotinic agonists, epibatidine and lobeline, and nicotinic antagonists, methyllycaconitine, alpha-bungarotoxin, and alpha-cobratoxin, markedly restricts the exchange of loop C amide protons, influencing both the rates and degrees of exchange. Solvent exposure of loop C and its protection by ligand suggest that in the apoprotein, loop C exhibits rapid fluctuations in an open conformation. Bound agonists restrict solvent exposure through loop closure, whereas the larger antagonists restrict solvent exposure largely through occlusion of solvent. Loop F, found on the complementary subunit surface at the interface, also reveals ligand selective changes in amide proton exchange rates. Agonists do not affect solvent accessibility of loop F, whereas certain antagonists cause subtle accessibility changes. These results reveal dynamic states and fluctuating movements in the vicinity of the binding site for unligated AChBP that can be influenced selectively by ligands.

Amino Acid Sequence↗

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

Thrombomodulin tightens the thrombin active site loops to promote protein C activation.

Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. Crystallographic investigations of the complex between thrombin and TMEGF456 did not show any changes in the thrombin active site. Therefore, research has focused recently on how TM may provide a docking site for the protein C substrate. Previous work, however, showed that when the thrombin active site was occupied with substrate analogues labeled with fluorophores, the fluorophores responded differently to active (TMEGF1-6) versus inactive (TMEGF56) fragments of TM. To investigate this further, we have carried out amide H/(2)H exchange experiments on thrombin in the presence of active (TMEGF45) and inactive (TMEGF56) fragments of TM. Both on-exchange and off-exchange experiments show changes in the thrombin active site loops, some of which are observed only when the active TM fragment is bound. These results are consistent with the previously observed fluorescence changes and point to a mechanism by which TM changes the thrombin substrate specificity in favor of protein C cleavage.

Amides↗

PubMed2005

The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family.

Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.

Algorithms↗

PubMed2005

Dynamics of the fragment of thrombomodulin containing the fourth and fifth epidermal growth factor-like domains correlate with function.

Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. The fourth and fifth EGF-like domains of TM together form the minimal fragment with anticoagulant cofactor activity. A short linker connects the fourth and fifth EGF-like domains of TM, and Met 388 in the middle of the linker interacts with both domains. Several different structures of TMEGF45 variants are now available, and these show that mutation of Met 388 alters the structure of the fifth domain, as well as the connectivity of the two domains. To probe this phenomenon more thoroughly, NMR backbone dynamics experiments have been carried out on the individual fourth and fifth domains as well as on the wild type, the Met 388 Leu mutant, and the variant in which Met 388 is oxidized. The results presented here show that changes at Met 388 cause significant changes in backbone dynamics in both the fourth and fifth EGF-like domains of TM. Backbone dynamics within the small loop of the fourth domain Tyr 358 correlate with anticoagulant cofactor activity. Backbone dynamics of the thrombin-binding residues Tyr 413 and Ile 414 are inversely correlated with thrombin binding. The preordering of the backbone of Tyr 413 and Ile 414 only occurs in the two-domain fragments, revealing a role for the fourth domain in thrombin binding as well as in anticoagulant cofactor activity.

Amino Acid Substitution↗

PubMed2005

Structural and functional consequences of methionine oxidation in thrombomodulin.

Thrombomodulin (TM) is an endothelial cell surface glycoprotein that is responsible for switching the catalytic activity of thrombin away from fibrinogen cleavage (pro-coagulant) and towards protein C cleavage (anticoagulant). Although TM is a large protein, only the fourth and fifth epidermal growth factor-like (EGF-like) domains are required for anticoagulant function. These two domains must work together, and the linker between the two domains contains a single methionine residue, Met 388. Oxidation of Met 388 is deleterious for TM activity. Structural studies, both X-ray and NMR, of wild type and variants at position 388 show that Met 388 provides a key linkage between the two domains. Oxidation of the methionine has consequences for the structure of the fifth domain, which binds to thrombin. Oxidation also appears to disrupt the interdomain contacts resulting in structural and dynamic changes. The functional consequences of oxidation of Met 388 include decreased anticoagulant activity. Oxidative stress from several causes is reflected in lower serum levels of activated protein C and a higher thrombotic tendency, and this is thought to be linked to the oxidation of Met 388 in TM. Thus, TM structure and function are altered in a subtle but functionally critical way upon oxidation of Met 388.

Amino Acid Sequence↗

PubMed2005

Measurement of solvent accessibility at protein-protein interfaces.

Methods are presented for monitoring solvent accessibility of protein-ligand and protein-protein interfaces. The kinetics of solvent accessibility at the protein-protein interface is monitored by amide hydrogen/deuterium (H/2H) exchange detected by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A straightforward theoretical analysis is presented for determining the concentration of a weakly binding ligand that is required for achieving a situation in which the receptor is essentially 100% bound, and this is verified by control experiments. We show that when the receptor is essentially 100% bound it is possible to distinguish amide exchange as a result of solvent accessibility at the interface from amide exchange caused by complex dissociation. Methods are also presented for the measurement of tightly bound complexes of large interactions such as antibody-antigen complexes. Quantitation of the number of amides sequestered at the interface can be related to the number of H2O molecules excluded from the interface.

Amides↗

PubMed2004

Two apolipoprotein E mimetic peptides, ApoE(130-149) and ApoE(141-155)2, bind to LRP1.

LRP1 is a cell surface receptor responsible for clearing some 30 known ligands. We have previously shown that each of the three complete LDL receptor-homology domains of the LRP1 extracellular domain (sLRPs) binds apoE-enriched beta-VLDL particles. Here we show that two peptides from the N-terminal receptor binding domain of apoE, which are known to elicit a number of different cellular responses, bind to LRP1. Solution binding assays show that the two peptides, apoE(130-149) and apoE(141-155)(2), interact with each of the sLRPs (2, 3, and 4). Each peptide was found to exhibit the same solution binding characteristics as apoE-enriched beta-VLDL particles. Surface plasmon resonance analyses of the sLRP-apoE peptide interaction show that both peptides bind the sLRPs with K(D) values in the 100 nM range, a value similar to the effective concentration required for observation of the cellular responses. Consistent with results from mutagenesis studies of binding of apoE to LDLR, apoE(130-149,Arg142Glu) bound with a K(D) similar to that of the wild-type sequence, while apoE(130-149,Lys143Glu) showed a 10-fold decrease in K(D). Each of the peptides bound heparin, and heparin competed for sLRP binding.

Amino Acid Sequence↗

PubMed2004

Allosteric changes in solvent accessibility observed in thrombin upon active site occupation.

The solvent accessibility of thrombin in its substrate-free and substrate-bound forms has been compared by amide hydrogen/deuterium (H/(2)H) exchange. The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) was used to simulate the substrate-bound form of thrombin. These studies were motivated by the lack of observed changes in the active site of thrombin in the crystal structure of the thrombin-thrombomodulin complex. This result appeared to contradict amide exchange studies on the thrombin-thrombomodulin complex that suggested subtle changes occur in the active site loops upon thrombomodulin binding. Our results show that two active site loops, residues 214-222 and residues 126-132, undergo decreases in solvent accessibility due to steric contacts with PPACK substrate. However, we also observe two regions outside the active site undergoing solvent protection upon substrate binding. The first region corresponds to anion binding exosite 1, and the second is a beta-strand-containing loop which runs through the core of the molecule and contains Trp141 which makes critical contacts with anion binding exosite 1. These results indicate two pathways of allosteric change that connect the active site to the distal anion binding exosite 1.

Allosteric Regulation↗

PubMed2004

Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles.

Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein-protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K(D)s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the DeltaG(bind) was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the DeltaG(bind) from electrostatic steering was assessed from the dependence of the k(a) on ionic strength. Release of solvent H(2)O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein-protein binding. The mAb binding is enthalpy driven and has a slow k(d). TM binding appears to be entropy driven and has a fast k(a). The favorable entropy of the thrombin-TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed.

Allosteric Regulation↗

PubMed2004

Biophysical characterization of the free IkappaBalpha ankyrin repeat domain in solution.

The crystal structure of IkappaBalpha in complex with the transcription factor, nuclear factor kappa-B (NF-kappaB) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IkappaBalpha (67-317) when it is not in complex with NF-kappaB, we have carried out circular dichroism (CD) spectroscopy, 8-anilino-1-napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. Ikappa Balpha unfolds with a T(m) of 42 degrees C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IkappaBalpha are discussed.

Amides↗

PubMed2003

All three LDL receptor homology regions of the LDL receptor-related protein bind multiple ligands.

The three complete human LDL receptor homology regions of the LDL receptor-related protein (sLRP2, sLRP3, and sLRP4) have been expressed in Pichia pastoris SMD1168 with constitutive coexpression of the receptor-associated protein (RAP). Each sLRP was purified to homogeneity after deglycosylation using a combination of anion-exchange and size exclusion chromatography. Mass spectrometry and N-terminal sequencing confirmed the identity of each fragment at purified yields of several milligrams per liter. Despite the large number of disulfide linkages and glycosylation sites in each LDL receptor homology region (sLRP), all were shown to be competent for binding to several LRP1 ligands. Each sLRP also bound human RAP, which is thought to be a generalized receptor antagonist, in solution-binding experiments. As expected, sLRP2 bound the receptor-binding domain of alpha(2)-macroglobulin (residues 1304-1451). All three sLRPs bound human apolipoprotein-enriched beta very low density lipoprotein, the canonical ligand for this receptor. All three sLRPs also bound lactoferrin and thrombin-protease nexin 1 complexes. Only sLRP4 bound thrombin-antithrombin III complexes. The results show that binding-competent LDL receptor homology regions (sLRPs) can be produced in high yield in P. pastoris and readily purified. Each sLRP has binding sites for multiple ligands, but not all ligand binding could be competed by RAP.

Amino Acid Sequence↗