Second-site suppressor mutations reveal connection between the drug-binding pocket and nucleotide-binding domain 1 of human P-glycoprotein (ABCB1).

Human P-glycoprotein (P-gp) or ABCB1 is overexpressed in many cancers and has been implicated in altering the bioavailability of chemotherapeutic drugs due to their efflux, resulting in the development of chemoresistance. To elucidate the mechanistic aspects and structure-function relationships of P-gp, we previously utilized a tyrosine (Y)-enriched P-gp mutant (15Y) and demonstrated that at least 15 conserved residues in the drug-binding pocket of P-gp are responsible for optimal substrate interaction and transport. To further understand the role of these 15 residues, two new mutants were generated, namely 6Y with the substitution of six residues (F72, F303, I306, F314, F336 and L339) with Y in transmembrane domain (TMD) 1 and 9Y with nine substitutions (F732, F759, F770, F938, F942, M949, L975, F983 and F994) in TMD2. Although both the mutants were expressed at normal levels at the cell surface, the 6Y mutant failed to transport all the tested substrates except Bodipy-verapamil, whereas the 9Y mutant effluxed all tested substrates in a manner very similar to that of the wild-type protein. Further mutational analysis revealed that two second-site mutations, one in intracellular helix (ICH) 4 (F916Y) and one in the Q loop of nucleotide-binding domain (NBD) 1 (F480Y) restored the transport function of 6Y. Additional biochemical data and comparative molecular dynamics simulations of the 6Y and 6Y+F916Y mutant indicate that the Q-loop of NBD1 of P-gp communicates with the substrate-binding sites in the transmembrane region through ICH4. This is the first evidence for the existence of second-site suppressors in human P-gp that allow recovery of the loss of transport function caused by primary mutations. Further study of such mutations could facilitate mapping of the communication pathway between the substrate-binding pocket and the NBDs of P-gp and possibly other ABC drug transporters.

Residues from Homologous Transmembrane Helices 4 and 10 Are Critical for P-Glycoprotein (ABCB1)-Mediated Drug Transport.

P-glycoprotein (P-gp, ABCB1) transports structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs, thus contributing to multidrug-resistant cancer. Cryo-EM structures of human P-gp revealed that TMHs 4 and 10 contribute to the formation of the drug-binding cavity and undergo conformational changes during drug transport. To assess the role of the conformational changes in TMH4 and TMH10 during drug transport, we generated two mutants (TMH4-7A and TMH10-7A), each containing seven alanine substitutions. Analysis of the drug efflux function of these mutants using 15 fluorescent substrates revealed that most of the substrates were transported, indicating that even seven mutations in an individual helix have no significant effect on transport function. We then designed the TMH4,10-14A mutant combining seven mutations in both TMHs 4 and 10. Interestingly, when the TMH4,10-14A mutant was tested with 15 substrates, there was no efflux observed for fourteen. The basal ATPase activity of the TMH4,10-14A mutant, similar to that of the WT protein, was inhibited by zosuquidar but was not stimulated by verapamil or rhodamine 6G. Molecular dynamics simulations indicated that the mutations cause TMHs 4 and 10 to pack tighter to their proximal helices, reducing their independent mobility. In aggregate, our findings demonstrate the critical role of the residues of homologous TMHs 4 and 10 for substrate transport, consistent with conformational changes observed in the structure of P-gp.

A compendium of co-regulated mitoribosomal proteins in pan-cancer uncovers collateral defective events in tumor malignancy.

Mitochondria are major organelles responsible for cellular energy and metabolism, and their dysfunction is tightly linked to cancer. The mitochondrial ribosome (mitoribosome) is a protein complex consisting of 82 mitoribosomal proteins (MRPs) encoded by nuclear genes and is essential for mitochondrial protein synthesis. However, their roles in tumorigenesis remain poorly understood. We performed pan-cancer analyses of 18,177 tumors representing 28 cancer types to determine somatic alterations of MRP genes as a genetic basis for tumorigenesis. We identified a set of 20 altered MRPs known to be involved in early assembly of the mitoribosome complex. We found that tumors with affected MRPs were associated with impaired mitochondrial functions and TP53 mutations accompanied by increased genomic instability and intra-tumor heterogeneity. MRP deletions were associated with poor survival. Our results reveal a key role for mitochondrial ribosome biogenesis in tumor malignancy across cancer types.

The amyloid concentric ß-barrel hypothesis: Models of amyloid beta 42 oligomers and annular protofibrils.

Amyloid beta (Aß) peptides are a major contributor to Alzheimer's disease. They occur in differing lengths, each of which forms a multitude of assembly types. The most toxic soluble oligomers are formed by Aß42; some of which have antiparallel ß-sheets. Previously, our group proposed molecular models of Aß42 hexamers in which the C-terminus third of the peptide (S3) forms an antiparallel 6-stranded ß-barrel that is surrounded by an antiparallel barrel formed by the more polar N-terminus (S1) and middle (S2) portions. These hexamers were proposed to act as seeds from which dodecamers, octadecamers, both smooth annular protofibrils (sAPFs) and beaded annular protofibrils (bAPFs), and transmembrane channels form. Since then, numerous aspects of our models have been supported by experimental findings. Recently, NMR-based structures have been proposed for Aß42 tetramers and octamers, and NMR studies have been reported for oligomers composed of ~32 monomers. Here we propose a range of concentric ß-barrel models and compare their dimensions to image-averaged electron micrographs of both bAPFs and sAPFs of Aß42. The smaller oligomers have 6, 8, 12, 16, and 18 monomers. These beads string together to form necklace-like bAPFs. These bAPRs gradually morph into sAPFs in which a S3 ß-barrel is shielded on one or both sides by ß-barrels formed from S1 and S2 segments.

Altering the Double-Stranded DNA Specificity of the bZIP Domain of Zta with Site-Directed Mutagenesis at N182.

Zta, the Epstein-Barr virus bZIP transcription factor (TF), binds both unmethylated and methylated double-stranded DNA (dsDNA) in a sequence-specific manner. We studied the contribution of a conserved asparagine (N182) to sequence-specific dsDNA binding to four types of dsDNA: (i) dsDNA with cytosine in both strands ((DNA(C|C)), (ii, iii) dsDNA with 5-methylcytosine (5mC, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand ((DNA(5mC|C) and DNA(5hmC|C)), and (iv) dsDNA with methylated cytosine in both strands in all CG dinucleotides ((DNA(5mCG)). We replaced asparagine with five similarly sized amino acids (glutamine (Q), serine (S), threonine (T), isoleucine (I), or valine (V)) and used protein binding microarrays to evaluate sequence-specific dsDNA binding. Zta preferentially binds the pseudo-palindrome TRE (AP1) motif (T-4G-3A-2G/C 0T2C3A4 ). Zta (N182Q) changes binding to A3 in only one half-site. Zta(N182S) changes binding to G3 in one or both halves of the motif. Zta(N182S) and Zta(N182Q) have 34- and 17-fold weaker median dsDNA binding, respectively. Zta(N182V) and Zta(N182I) have increased binding to dsDNA(5mC|C). Molecular dynamics simulations rationalize some of these results, identifying hydrogen bonds between glutamine and A3 , but do not reveal why serine preferentially binds G3 , suggesting that entropic interactions may mediate this new binding specificity.

The amyloid concentric ß-barrel hypothesis: Models of synuclein oligomers, annular protofibrils, lipoproteins, and transmembrane channels.

Amyloid beta (Aß of Alzheimer's disease) and α-synuclein (α-Syn of Parkinson's disease) form large fibrils. Evidence is increasing however that much smaller oligomers are more toxic and that these oligomers can form transmembrane ion channels. We have proposed previously that Aß42 oligomers, annular protofibrils, and ion channels adopt concentric ß-barrel molecular structures. Here we extend that hypothesis to the superfamily of α, ß, and γ-synucleins. Our models of numerous synuclein oligomers, annular protofibrils, tubular protofibrils, lipoproteins, and ion channels were developed to be consistent with sizes, shapes, molecular weights, and secondary structures of assemblies as determined by electron microscopy and other studies. The models have the following features: (1) all subunits have identical structures and interactions; (2) they are consistent with conventional ß-barrel theory; (3) the distance between walls of adjacent ß-barrels is between 0.6 and 1.2 nm; (4) hydrogen bonds, salt bridges, interactions among aromatic side-chains, burial and tight packing of hydrophobic side-chains, and aqueous solvent exposure of hydrophilic side-chains are relatively optimal; and (5) residues that are identical among distantly related homologous proteins cluster in the interior of most oligomers whereas residues that are hypervariable are exposed on protein surfaces. Atomic scale models of some assemblies were developed.

Temperature Dependence of Hydrophobic and Hydrophilic Forces and Interactions.

Molecular dynamics simulations are used to compare the forces and Gibbs free energies associated with bringing small hydrophobic and hydrophilic solutes together in an aqueous solution at different temperatures between 280 and 360 °K. For the hydrophilic solutes, different relative orientations are used to distinguish between direct, intersolute hydrogen bonds (Hbond) and solutes simultaneously hydrogen bonding to a solvent water bridge. Interestingly, the temperature dependence of the hydrophobic and directly hydrogen bonding solutes turns out to be opposite to that of the bridged hydrophilic solutes, with the ΔG becoming more negative for the former and less negative for the latter with increasing temperature. Dissection of the free energy curves into enthalpy and entropy contributions, and further separation of the enthalpy term into solute-solute, solute-solvent, and solvent-solvent components provides insight into the physical molecular causes for the distinctive thermodynamic results. Finally, it is reasoned how the opposite temperature dependencies of the two types of hydrophilic interactions provide a rationale for the cold denaturation of proteins.

REL Domain of NFATc2 Binding to Five Types of DNA Using Protein Binding Microarrays.

NFATc2 is a DNA binding protein in the Rel family transcription factors, which binds a CGGAA motif better when both cytosines in the CG dinucleotide are methylated. Using protein binding microarrays (PBMs), we examined the DNA binding of NFATc2 to three additional types of DNA: single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with either 5-methylcytosine (5mC, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and a cytosine in the second strand. ATTTCCAC, the complement of the core GGAA motif, is better bound as ssDNA compared to dsDNA. dsDNA containing the 5-mer CGGAA with either 5mC or 5hmC in one DNA strand is bound stronger than CGGAA. In contrast, the reverse complement TTCCG is bound weaker when it contains 5mC. Analysis of the available NFATc2:dsDNA complexes rationalizes these PBM data.

Does the ATP-bound EQ mutant reflect the pre- or post-ATP hydrolysis state in the catalytic cycle of human P-glycoprotein (ABCB1)?

P-glycoprotein (P-gp, ABCB1) is an ABC transporter associated with the development of multidrug resistance to chemotherapy. During its catalytic cycle, P-gp undergoes significant conformational changes. Recently, atomic structures of some of these conformations have been resolved using cryo-electron microscopy. The ATP hydrolysis-defective mutant of the catalytic glutamate residue of the Walker B motif (E556Q/E1201Q) has been used to determine the structure of the ATP-bound inward-closed conformation of P-gp. Here, we show that this mutant does not appear to undergo the same steps as wild-type P-gp. We discuss conformational differences in the EQ mutant that may lead to a better understanding of the catalytic cycle of P-gp and propose that additional structural studies with wild-type P-gp are required.

Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein.

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

The structure-activity profile of mercaptobenzamides' anti-HIV activity suggests that thermodynamics of metabolism is more important than binding affinity to the target.

Mercaptobenzamide thioesters and thioethers are chemically simple HIV-1 maturation inhibitors with a unique mechanism of action, low toxicity, and a high barrier to viral resistance. A structure-activity relationship (SAR) profile based on 39 mercaptobenzamide prodrug analogs exposed divergent activity/toxicity roles for the internal and terminal amides. To probe the relationship between antiviral activity and toxicity, we generated an improved computational model for the binding of mercaptobenzamide thioesters (SAMTs) to the HIV-1 NCp7 C-terminal zinc finger, revealing the presence of a second low-energy binding orientation, hitherto undisclosed. Finally, using NMR-derived thiol-thioester exchange equilibrium constants, we propose that thermodynamics plays a role in determining the antiviral activity observed in the SAR profile.

Evidence for the Interaction of A3 Adenosine Receptor Agonists at the Drug-Binding Site(s) of Human P-glycoprotein (ABCB1).

P-glycoprotein (P-gp) is a multidrug transporter that is expressed on the luminal surface of epithelial cells in the kidney, intestine, bile-canalicular membrane in the liver, blood-brain barrier, and adrenal gland. This transporter uses energy of ATP hydrolysis to efflux from cells a variety of structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs. In this regard, understanding the interaction with P-gp of drug entities in development is important and highly recommended in current US Food and Drug Administration guidelines. Here we tested the P-gp interaction of some A3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Biochemical assays of the ATPase activity of P-gp and by photolabeling P-gp with its transport substrate [125I]-iodoarylazidoprazosin led to the identification of rigidified (N)-methanocarba nucleosides (i.e., compound 3 as a stimulator and compound 8 as a partial inhibitor of P-gp ATPase activity). Compound 8 significantly inhibited boron-dipyrromethene (BODIPY)-verapamil transport mediated by human P-gp (IC50 2.4 ± 0.6 µM); however, the BODIPY-conjugated derivative of 8 (compound 24) was not transported by P-gp. In silico docking of compounds 3 and 8 was performed using the recently solved atomic structure of paclitaxel (Taxol)-bound human P-gp. Molecular modeling studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as Taxol. Thus, this study indicates that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. SIGNIFICANCE STATEMENT: Certain A3 adenosine receptor agonists are being developed for the treatment of chronic diseases. The goal of this study was to test the interaction of these agonists with the human multidrug resistance-linked transporter P-glycoprotein (P-gp). ATPase and photolabeling assays demonstrated that compounds with rigidified (N)-methanocarba nucleosides inhibit the activity of P-gp; however, a fluorescent derivative of one of the compounds was not transported by P-gp. Furthermore, molecular docking studies revealed that the binding site for these compounds overlaps with the site for paclitaxel in the drug-binding pocket. These results suggest that nucleoside derivatives, depending on structural functionalization, can modulate the function of P-gp.

The bZIP mutant CEBPB (V285A) has sequence specific DNA binding propensities similar to CREB1.

The bZIP homodimers CEBPB and CREB1 bind DNA containing methylated cytosines differently. CREB1 binds stronger to the C/EBP half-site GCAA when the cytosine is methylated. For CEBPB, methylation of the same cytosine does not affect DNA binding. The X-ray structure of CREB1 binding the half site GTCA identifies an alanine in the DNA binding region interacting with the methyl group of T, structurally analogous to the methyl group of methylated C. This alanine is replaced with a valine in CEBPB. To explore the contribution of this amino acid to binding with methylated cytosine of the GCAA half-site, we made the reciprocal mutants CEBPB(V285A) and CREB1(A297V) and used protein binding microarrays (PBM) to examine binding to four types of double-stranded DNA (dsDNA): 1) DNA with cytosine in both strands (DNA(C|C)), 2) DNA with 5-methylcytosine (M) in one strand and cytosine in the second strand (DNA(M|C)), 3) DNA with 5-hydroxymethylcytosine (H) in one strand and cytosine in the second strand (DNA(H|C)), and 4) DNA with both cytosines in all CG dinucleotides containing 5-methylcytosine (DNA(5mCG)). When binding to DNA(C|C), CEBPB (V285A) preferentially binds the CRE consensus motif (TGACGTCA), similar to CREB1. The reciprocal mutant, CREB1(A297V) binds DNA with some similarity to CEBPB, with strongest binding to the methylated PAR site 8-mer TTACGTAA. These data demonstrate that V285 residue inhibits CEBPB binding to methylated cytosine of the GCAA half-site.

Resistance to Second-Generation HIV-1 Maturation Inhibitors.

A betulinic acid-based compound, bevirimat (BVM), inhibits HIV-1 maturation by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. Previous studies showed that mutations conferring resistance to BVM cluster around the CA-SP1 cleavage site. Single amino acid polymorphisms in the SP1 region of Gag and the C terminus of CA reduced HIV-1 susceptibility to BVM, leading to the discontinuation of BVM's clinical development. We recently reported a series of "second-generation" BVM analogs that display markedly improved potency and breadth of activity relative to the parent molecule. Here, we demonstrate that viral clones bearing BVM resistance mutations near the C terminus of CA are potently inhibited by second-generation BVM analogs. We performed de novo selection experiments to identify mutations that confer resistance to these novel compounds. Selection experiments with subtype B HIV-1 identified an Ala-to-Val mutation at SP1 residue 1 and a Pro-to-Ala mutation at CA residue 157 within the major homology region (MHR). In selection experiments with subtype C HIV-1, we identified mutations at CA residue 230 (CA-V230M) and SP1 residue 1 (SP1-A1V), residue 5 (SP1-S5N), and residue 10 (SP1-G10R). The positions at which resistance mutations arose are highly conserved across multiple subtypes of HIV-1. We demonstrate that the mutations confer modest to high-level maturation inhibitor resistance. In most cases, resistance was not associated with a detectable increase in the kinetics of CA-SP1 processing. These results identify mutations that confer resistance to second-generation maturation inhibitors and provide novel insights into the mechanism of resistance.IMPORTANCE HIV-1 maturation inhibitors are a class of small-molecule compounds that block a late step in the viral protease-mediated processing of the Gag polyprotein precursor, the viral protein responsible for the formation of virus particles. The first-in-class HIV-1 maturation inhibitor bevirimat was highly effective in blocking HIV-1 replication, but its activity was compromised by naturally occurring sequence polymorphisms within Gag. Recently developed bevirimat analogs, referred to as "second-generation" maturation inhibitors, overcome this issue. To understand more about how these second-generation compounds block HIV-1 maturation, here we selected for HIV-1 mutants that are resistant to these compounds. Selections were performed in the context of two different subtypes of HIV-1. We identified a small set of mutations at highly conserved positions within the capsid and spacer peptide 1 domains of Gag that confer resistance. Identification and analysis of these maturation inhibitor-resistant mutants provide insights into the mechanisms of resistance to these compounds.

Mapping discontinuous epitopes for MRK-16, UIC2 and 4E3 antibodies to extracellular loops 1 and 4 of human P-glycoprotein.

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is associated with the development of multidrug resistance in cancer cells. Antibody-mediated blockade of human P-gp activity has been shown to overcome drug resistance by re-sensitizing resistant cancer cells to anticancer drugs. Despite the potential clinical application of this finding, the epitopes of the three human P-gp-specific monoclonal antibodies MRK-16, UIC2 and 4E3, which bind to the extracellular loops (ECLs) have not yet been mapped. By generating human-mouse P-gp chimeras, we mapped the epitopes of these antibodies to ECLs 1 and 4. We then identified key amino acids in these regions by replacing mouse residues with homologous human P-gp residues to recover binding of antibodies to the mouse P-gp. We found that changing a total of ten residues, five each in ECL1 and ECL4, was sufficient to recover binding of both MRK-16 and 4E3 antibodies, suggesting a common epitope. However, recovery of the conformation-sensitive UIC2 epitope required replacement of thirteen residues in ECL1 and the same five residues replaced in the ECL4 for MRK-16 and 4E3 binding. These results demonstrate that discontinuous epitopes for MRK-16, UIC2 and 4E3 are located in the same regions of ECL1 and 4 of the multidrug transporter.

Replacing C189 in the bZIP domain of Zta with S, T, V, or A changes DNA binding specificity to four types of double-stranded DNA.

Zta is a bZIP transcription factor (TF) in the Epstein-Barr virus that binds unmethylated and methylated DNA sequences. Substitution of cysteine 189 of Zta to serine (Zta(C189S)) results in a virus that is unable to execute the lytic cycle, which was attributed to a change in binding to methylated DNA sequences. To learn more about the role of this position in defining sequence-specific DNA binding, we mutated cysteine 189 to four other amino acids, producing Zta(C189S), Zta(C189T), Zta(C189A), and Zta(C189V) mutants. Zta and mutants were used in protein binding microarray (PBM) experiments to evaluate sequence-specific DNA binding to four types of double-stranded DNA (dsDNA): 1) with cytosine in both strands (DNA(C|C)), 2) with 5-methylcytosine (5mC) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). Zta(C189S) and Zta(C189T) bound the TRE (AP-1) motif (TGAG/CTCA) more strongly than wild-type Zta, while binding to other sequences, including the C/EBP half site GCAA was reduced. Binding of Zta(C189S) and Zta(C189T) to DNA containing modified cytosines (DNA(5mC|C), DNA(5hmC|C), and DNA(5mCG)) was reduced compared to Zta. Zta(C189A) and Zta(C189V) had higher non-specific binding to all four types of DNA. Our data suggests that position C189 in Zta impacts sequence-specific binding to DNA containing modified and unmodified cytosine.

A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity.

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

The Epstein-Barr Virus B-ZIP Protein Zta Recognizes Specific DNA Sequences Containing 5-Methylcytosine and 5-Hydroxymethylcytosine.

The Epstein-Barr virus (EBV) B-ZIP transcription factor Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the sequence specific DNA binding of Zta to four kinds of double-stranded DNA (dsDNA): (1) DNA containing cytosine in both strands, (2) DNA with 5-methylcytosine (5mC) in one strand and cytosine in the second strand, (3) DNA with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand, and (4) DNA in which both cytosines in all CG dinucleotides contain 5mC. We compared these data to PBM data for three additional B-ZIP proteins (CREB1 and CEBPB homodimers and cJun|cFos heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M = 5mC), were preferentially bound. 5mC inhibits binding of Zta to both TRE motif half-sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer more strongly bind the C/EBP half-site tetranucleotide GCAA when it contains 5mC. Zta also binds dsDNA sequences containing 5hmC in one strand, although the effect is less dramatic than that observed for 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, uncovering the potential for discovery of new viral and host regulatory programs controlled by EBV.

Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry.

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange-mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30-300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192-200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.

Hydrophobic-hydrophilic forces in protein folding.

The process of protein folding is obviously driven by forces exerted on the atoms of the amino-acid chain. These forces arise from interactions with other parts of the protein itself (direct forces), as well as from interactions with the solvent (solvent-induced forces). We present a statistical-mechanical formalism that describes both these direct and indirect, solvent-induced thermodynamic forces on groups of the protein. We focus on 2 kinds of protein groups, commonly referred to as hydrophobic and hydrophilic. Analysis of this result leads to the conclusion that the forces on hydrophilic groups are in general stronger than on hydrophobic groups. This is then tested and verified by a series of molecular dynamics simulations, examining both hydrophobic alkanes of different sizes and hydrophilic moieties represented by polar-neutral hydroxyl groups. The magnitude of the force on assemblies of hydrophilic groups is dependent on their relative orientation: with 2 to 4 times larger forces on groups that are able to form one or more direct hydrogen bonds.