Mark S. Searle - Cochemist

Ligand selectivity in stabilising tandem parallel folded G-quadruplex motifs in human telomeric DNA sequences

Co-reporter:Alex R. O. Cousins, Dougal Ritson, Pallavi Sharma, Malcolm F. G. Stevens, John E. Moses and Mark S. Searle

Chemical Communications 2014 vol. 50(Issue 96) pp:15202-15205

Publication Date(Web):23 Oct 2014

DOI:10.1039/C4CC07487D

Biophysical studies of ligand interactions with three human telomeric repeat sequences (d(AGGG(TTAGGG)n, n = 3, 7 and 11)) show that an oxazole-based ‘click’ ligand, which induces parallel folded quadruplexes, preferentially stabilises longer telomeric repeats providing evidence for selectivity in binding at the interface between tandem quadruplex motifs.

Enthalpy/Entropy Compensation Effects from Cavity Desolvation Underpin Broad Ligand Binding Selectivity for Rat Odorant Binding Protein 3

Co-reporter:Katherine L. Portman, Jed Long, Stephen Carr, Loïc Briand, Donald J. Winzor, Mark S. Searle, and David J. Scott

Biochemistry 2014 Volume 53(Issue 14) pp:2371-2379

Publication Date(Web):March 26, 2014

DOI:10.1021/bi5002344

Evolution has produced proteins with exquisite ligand binding specificity, and manipulating this effect has been the basis for much of modern rational drug design. However, there are general classes of proteins with broader ligand selectivity linked to function, the origin of which is poorly understood. The odorant binding proteins (OBPs) sequester volatile molecules for transportation to the olfactory receptors. Rat OBP3, which we characterize by X-ray crystallography and NMR, binds a homologous series of aliphatic γ-lactones within its aromatic-rich hydrophobic pocket with remarkably little variation in affinity but extensive enthalpy/entropy compensation effects. We show that the binding energetics are modulated by two desolvation processes with quite different thermodynamic signatures. Ligand desolvation follows the classical hydrophobic effect; however, cavity desolvation is consistent with the liberation of “high energy” water molecules back into bulk solvent with a strong, but compensated, enthalpic contribution, which together underpin the origins of broad ligand binding selectivity.

Independent Interactions of Ubiquitin-Binding Domains in a Ubiquitin-Mediated Ternary Complex

Co-reporter:Thomas P. Garner, Joanna Strachan, Elizabeth C. Shedden, Jed E. Long, James R. Cavey, Barry Shaw, Robert Layfield, and Mark S. Searle

Biochemistry 2011 Volume 50(Issue 42) pp:

Publication Date(Web):September 16, 2011

DOI:10.1021/bi201137e

Ubiquitin (Ub) modifications are transduced by receptor proteins that use Ub-binding domains (UBDs) to recognize distinct interaction faces on the Ub surface. We report the nuclear magnetic resonance (NMR) solution structures of the A20-like zinc finger (A20 Znf) UBD of the Ub receptor ZNF216, and its complex with Ub, and show that the binding surface on Ub centered on Asp58 leaves the canonical hydrophobic Ile44 patch free to participate in additional interactions. We have modeled ternary complexes of the different families of UBDs and show that while many are expected to bind competitively to the same Ile44 surface or show steric incompatibility, other combinations (in particular, those involving the A20 Znf domain) are consistent with a single Ub moiety simultaneously participating in multiple interactions with different UBDs. We subsequently demonstrate by NMR that the A20 Znf domain of ZNF216 and the UBA domain of the p62 protein (an Ile44-binding UBD), which function in the same biological pathways, are able to form such a Ub-mediated ternary complex through independent interactions with a single Ub. This work supports an emerging concept of Ub acting as a scaffold to mediate multiprotein complex assembly.

Impact of p62/SQSTM1 UBA Domain Mutations Linked to Paget’s Disease of Bone on Ubiquitin Recognition

Co-reporter:Thomas P. Garner, Jed Long, Robert Layfield, and Mark S. Searle

Biochemistry 2011 Volume 50(Issue 21) pp:

Publication Date(Web):April 25, 2011

DOI:10.1021/bi200079n

The scaffold protein p62/SQSTM1 acts as a hub in regulating a diverse range of signaling pathways which are dependent upon a functional ubiquitin-binding C-terminal UBA domain. Mutations linked to Paget’s disease of bone (PDB) commonly cluster within the UBA domain. The p62 UBA domain is unique in forming a highly stable dimer which regulates ubiquitin recognition by using overlapping surface patches in both dimerization and ubiquitin binding, making the two association events competitive. NMR structural analysis and biophysical methods show that some PDB mutations modulated the ubiquitin binding affinity by both direct and indirect mechanisms that affect UBA structural integrity, dimer stability, and contacts at the UBA–ubiquitin interface. In other cases, common PDB mutations (P392L in particular) result in no significant change in ubiquitin binding affinity for the UBA domain in isolation; however, all PDB UBA mutations lead to loss of function with respect to ubiquitin binding in the context of full-length p62, suggesting a more complex underlying mechanism.

Structural Insights into the Two Sequential Folding Transition States of the PB1 Domain of NBR1 from Φ Value Analysis and Biased Molecular Dynamics Simulations

Co-reporter:Ping Chen, Clare-Louise Evans, Jonathan D. Hirst, and Mark S. Searle

Biochemistry 2011 Volume 50(Issue 1) pp:

Publication Date(Web):December 1, 2010

DOI:10.1021/bi1016793

The PB1 domain of NBR1 folds via a single pathway mechanism involving two sequential energy barriers separated by a high-energy intermediate. The structural ensemble representing each of the two transition states (TS1 and TS2) has been calculated using experimental Φ values and biased molecular dynamics simulations. Both TS1 and TS2 represent compact states (βTS1 = 0.71, and βTS2 = 0.93) but are defined by quite different distributions of Φ values, degrees of structural heterogeneity, and nativelike secondary structure. TS1 forms a heterogeneous ensemble of dynamic structures, representing a global collapse of the polypeptide chain around a set of weak nativelike contacts. In contrast, TS2 has a high proportion of nativelike secondary structure, which is reflected in an extensive distribution of high Φ values. Two snapshots along the folding pathway of the PB1 domain reveal insights into the malleability, the solvent accessibility, and the timing of nativelike core packing that stabilizes the folded state.

Occurrence of a Quadruplex Motif in a Unique Insert within Exon C of the Bovine Estrogen Receptor α Gene (ESR1)

Co-reporter:Kamila Derecka, Graham D. Balkwill, Thomas P. Garner, Charlie Hodgman, Anthony P. F. Flint and Mark S. Searle

Biochemistry 2010 Volume 49(Issue 35) pp:

Publication Date(Web):July 26, 2010

DOI:10.1021/bi100804f

The 5′ end of exon C of the bovine estrogen receptor α gene (bov-ESR1) includes a unique G-rich insert, not found in other closely related mammalian genes, which lies close to both a double E-box transcription factor binding site and the site of a single nucleotide (G/A) polymorphism. Biophysical studies, using CD and UV absorbance measurements, show that this 22 base insert leads to the formation of a family of stable G-quadruplex structures which are unaffected by the G/A polymorphism. Multiplex PCR shows that the region including the G-quadruplex is transcribed into RNA, and studies with a synthetic RNA transcript sequence demonstrated formation of a highly stable parallel-folded quadruplex structure. Luciferase reporter constructs demonstrate that the G-rich sequence reduces rates of translation when present in the 5′-UTR of mRNA transcripts. Mutations (GGG to AAA) that destabilize the quadruplex lead to a 15-fold enhancement of translational efficiency, suggesting that a possible biological role of the insert in exon C of the bov-ESR1 is to regulate translation of this exon.

Selectivity of small molecule ligands for parallel and anti-parallel DNA G-quadruplex structures

Co-reporter:Thomas P. Garner, Huw E. L. Williams, Katarzyna I. Gluszyk, Stephen Roe, Neil J. Oldham, Malcolm F. G. Stevens, John E. Moses and Mark S. Searle

Organic & Biomolecular Chemistry 2009 vol. 7(Issue 20) pp:4194-4200

Publication Date(Web):14 Aug 2009

DOI:10.1039/B910505K

We report CD, ESI-MS and molecular modelling studies of ligand binding interactions with DNA quadruplex structures derived from the human telomeric repeat sequence (h-Tel) and the proto-oncogenic c-kit promoter sequence. These sequences form anti-parallel (both 2 + 2 and 3 + 1) and parallel conformations, respectively, and demonstrate distinctively different degrees of structural plasticity in binding ligands. With h-Tel, we show that an extended heteroaromatic 1,4-triazole (TRZ), designed to exploit π-stacking interactions and groove-specific contacts, shows some selectivity for parallel folds, however, the polycyclic fluorinated acridinium cation (RHPS4), which is a similarly potent telomerase inhibitor, shows selectivity for anti-parallel conformations implicating favourable interactions with lateral and diagonal loops. In contrast, the unique c-kit parallel-stranded quadruplex shows none of the structural plasticity of h-Tel with either ligand. We show by quantitative ESI-MS analysis that both sequences are able to bind a ligand on either end of the quadruplex. In the case of h-Tel the two sites have similar affinities, however, in the case of the c-kit quadruplex the affinities of the two sites are different and ligand-dependent. We demonstrate that two different small molecule architectures result in significant differences in selectivity for parallel and anti-parallel quadruplex structures that may guide quadruplex targeted drug-design.

Repression of Translation of Human Estrogen Receptor α by G-Quadruplex Formation

Co-reporter:Graham D. Balkwill, Kamila Derecka, Thomas P. Garner, Charlie Hodgman, Anthony P. F. Flint and Mark S. Searle

Biochemistry 2009 Volume 48(Issue 48) pp:

Publication Date(Web):October 27, 2009

DOI:10.1021/bi901420k

Tissue-specific expression of the human estrogen receptor α gene (ESR1) is achieved through multiple promoter sequences resulting in various mRNA transcripts encoding a common protein but differing in their 5′-untranslated region (5′-UTR). Many cancers are estrogen-sensitive with neoplastic growth stimulated through the estrogen receptor, a transcription factor that regulates developmental genes. We demonstrate that the human ESR1 gene is rich in potential quadruplex-forming sequences with 3 of 20 identified within exonic regions. In particular, we show using CD, UV, and NMR spectroscopy that a stable DNA G-quadruplex motif is formed within the exon C gene sequence. This motif, which PCR shows is transcribed in normal and neoplastic endometrium and in MCF-7 cells, forms a stable RNA quadruplex demonstrable by CD and UV analysis. Cloning the exon C G-quadruplex sequence upstream of a luciferase reporter gene caused a 6-fold reduction of enzymatic activity compared to a mutant sequence. We conclude that the exon C G-quadruplex motif is present in the 5′-UTR of the mRNA transcript, where it modulates the efficiency of translation.

Mechanism of Ligand-Induced Folding of a Natively Unfolded Helixless Variant of Rabbit I-BABP

Co-reporter:Anita M. Rea, Victoria Thurston and Mark S. Searle

Biochemistry 2009 Volume 48(Issue 31) pp:

Publication Date(Web):July 6, 2009

DOI:10.1021/bi900805s

Substitution of the helix−turn−helix capping motif (residues 9−35) of rabbit I-BABP with a flexible Gly-Gly-Ser-Gly linker results in the loss of stabilizing hydrophobic contacts and renders the β-clamshell structure of this steroidal bile acid transport protein unfolded. However, in the presence of a bile acid ligand, we observe strong coupling between binding and folding, resulting in an enthalpy-driven high-affinity interaction (KA ∼ 4 × 105 M−1) that “rescues” the native state. We investigate the mechanism of induced folding using fluorescence stopped-flow kinetic measurements to distinguish between conformational selection and induced-fit models. We observe both ligand-dependent and -independent kinetic phases which, together with their relative amplitudes, we attribute to an induced-fit “fly casting” type of model in which transient encounter complexes between the ligand and the extended polypeptide chain may act as nucleation sites for folding. An initial fast ligand-dependent kinetic process appears to be consistent with formation of a hydrophobically collapsed intermediate state which slowly rearranges to a nativelike β-clamshell structure. We show that the intermediate forms at a rate 1000 times slower than the rate of ligand association with wild-type I-BABP, reflecting the large configurational entropic barrier to the coupled binding and folding steps of Δα-I-BABP. We have provided mechanistic insights into how natively disordered states, now commonly identified in biology, may fold on binding a target substrate or ligand.

Aromatic Residues Engineered into the β-Turn Nucleation Site of Ubiquitin Lead to a Complex Folding Landscape, Non-Native Side-Chain Interactions, and Kinetic Traps†

Co-reporter:Anita M. Rea, Emma R. Simpson, Jill K. Meldrum, Huw E. L. Williams and Mark S. Searle

Biochemistry 2008 Volume 47(Issue 48) pp:12910-12922

Publication Date(Web):November 7, 2008

DOI:10.1021/bi801330r

The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the β-turn region of the N-terminal β-hairpin, using both the native G-bulged type I turn sequence (TXTGK) as well as an engineered 2:2 XNGK type I′ turn sequence. The N-terminal β-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I′ turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U→I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized β-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.

Helix Mutations Stabilize a Late Productive Intermediate on the Folding Pathway of Ubiquitin

Co-reporter:Anita M. Rea, Emma R. Simpson, Maria D. Crespo and Mark S. Searle

Biochemistry 2008 Volume 47(Issue 31) pp:

Publication Date(Web):July 11, 2008

DOI:10.1021/bi800722d

We have investigated the relative placement of rate-limiting energy barriers and the role of productive or obstructive intermediates on the folding pathway of yeast wild-type ubiquitin (wt-Ub) containing the F45W mutation. To manipulate the folding barriers, we have designed a family of mutants in which stabilizing substitutions have been introduced incrementally on the solvent-exposed surface of the main α-helix (residues 23−34), which has a low intrinsic helical propensity in the native sequence. Although the U → I and I → N transitions are not clearly delineated in the kinetics of wt-Ub, we show that an intermediate becomes highly populated and more clearly resolved as the predicted stability of the helix increases. The observed acceleration in the rate of folding correlates with helix stability and is consistent with the I-state representing a productive rather than misfolded state. A Leffler analysis of the effects on kinetics of changes in stability within the family of helix mutants results in a biphasic correlation in both the refolding and unfolding rates that suggest a shift from a nucleation−condensation mechanism (weakly stabilized helix) toward a diffusion−collision model (highly stabilized helix). Through the introduction of helix-stabilizing mutations, we are able to engineer a well-resolved I-state on the folding pathway of ubiquitin which is likely to be structurally distinct from that which is only weakly populated on the folding pathway of wild-type ubiquitin.

Structure and folding dynamics of a DNA hairpin with a stabilising d(GNA) trinucleotide loop: influence of base pair mis-matches and point mutations on conformational equilibria

Co-reporter:Graham D. Balkwill, Huw E. L. Williams and Mark S. Searle

Organic & Biomolecular Chemistry 2007 vol. 5(Issue 5) pp:832-839

Publication Date(Web):25 Jan 2007

DOI:10.1039/B616820E

Hairpins are known to play specific roles in DNA– and RNA–protein recognition. Various disease states are thought to originate from the ill-timed formation of a hairpin loop during transcription, particularly in the context of triplet repeats which are associated with myotonic dystrophy, fragile X syndrome and other genetic disorders. An understanding of nucleic acid folding mechanisms requires a detailed appreciation of the timescales of these local folding events, a characterisation of the conformational equilibria that exist in solution and the influence of point mutations on the relative stabilities of the different species. We investigate using NMR and CD spectroscopy the structure and dynamics of a DNA hairpin containing a highly stabilising cGNAg loop. The single-stranded 13-mer 5′-d(GCTACGNAGTCGC) with N = T folds to form a hairpin structure which accommodates a C–T mis-matched base pair within the double-stranded stem region. The hairpin is in equilibrium with a double-stranded duplex form with the mixture of two interconverting conformations in slow exchange on the NMR timescale (1–2 s–1 at 308 K). We are able to characterise the dynamics of the interconversion process by NMR magnetisation transfer and by CD stopped-flow kinetic experiments. The latter shows that the hairpin folds too rapidly to detect by this method (>500 s–1) and forms in a “kinetic overshoot” followed by a much slower equilibration to a mixture of conformations (∼0.13 s–1 at 298 K). A point mutation that converts the GTA to a GAA loop sequence destabilises the intermolecular duplex structure and enables us to unambiguously assign the various dynamic processes that are taking place.

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein

Co-reporter:Nikolaos Kouvatsos, Jill K. Meldrum, Mark S. Searle and Neil R. Thomas

Chemical Communications 2006 (Issue 44) pp:4623-4625

Publication Date(Web):27 Sep 2006

DOI:10.1039/B610130E

We have engineered a variant of the β-clam shell protein ILBP which lacks the α-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like β-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

Engineering Enhanced Protein Stability through β-Turn Optimization: Insights for the Design of Stable Peptide β-Hairpin Systems

Co-reporter:Emma R. Simpson;Jill K. Meldrum;Roger Bofill Dr.;Maria D. Crespo Dr.;Elizabeth Holmes

Angewandte Chemie 2005 Volume 117(Issue 31) pp:

Publication Date(Web):6 JUL 2005

DOI:10.1002/ange.200500577

Ein guter Dienst: Haarnadelschleifen sind eine wichtige Komponente von Proteinstrukturen und Ausgangspunkte für die β-Haarnadel-Faltung. Mit Ubiquitin als „Wirt“-System (siehe Struktur) wurden Protein-Engineering-Methoden verwendet, um den Energiebeitrag von Typ-I′-Haarnadelschleifen zur Stabilität von Proteinen zu bestimmen, wobei das Ziel war, das rationale Design von Modell-Peptidsystemen zu optimieren.

Engineering Enhanced Protein Stability through β-Turn Optimization: Insights for the Design of Stable Peptide β-Hairpin Systems

Co-reporter:Emma R. Simpson;Jill K. Meldrum;Roger Bofill Dr.;Maria D. Crespo Dr.;Elizabeth Holmes

Angewandte Chemie International Edition 2005 Volume 44(Issue 31) pp:

Publication Date(Web):6 JUL 2005

DOI:10.1002/anie.200500577

One good turn: β-Turns are an important component of protein structure and nucleation sites for β-hairpin folding. With ubiquitin as a “host” system (see structure), protein engineering methods have been used to determine the energetic contribution of type I′ β-turns to the stability of proteins, with a view to optimizing the rational design of model peptide systems.

Incremental Contribution to Protein Stability from a β Hairpin “Finger”: Limits on the Stability of Designed β Hairpin Peptides

Co-reporter:Mark S. Searle ;Geoffrey W. Platt Dr.;Roger Bofill Dr.;Stephen A. Simpson;Barbara Ciani Dr.

Angewandte Chemie International Edition 2004 Volume 43(Issue 15) pp:

Publication Date(Web):30 MAR 2004

DOI:10.1002/anie.200352955

Back to the fold: A new method to determining the stability of a β hairpin is described. A β hairpin forming sequence (β4) is introduced into native ubiquitin (see structure) enabling the contribution to protein stability of this structural motif to be estimated. This data provides both an upper limit on stability for autonomously folding β hairpins, and a spectroscopic reference state for estimating the stability of related peptides in solution.

Incremental Contribution to Protein Stability from a β Hairpin “Finger”: Limits on the Stability of Designed β Hairpin Peptides

Co-reporter:Mark S. Searle ;Geoffrey W. Platt Dr.;Roger Bofill Dr.;Stephen A. Simpson;Barbara Ciani Dr.

Angewandte Chemie 2004 Volume 116(Issue 15) pp:

Publication Date(Web):30 MAR 2004

DOI:10.1002/ange.200352955

Haarig: Eine Methode zur Bestimmung der Stabilität einer β-Haarnadelstruktur wird beschrieben. Eine β-Haarnadel bildende Sequenz (β4) wird in natives Ubiquitin (siehe Struktur) eingebaut, wodurch der Protein stabilisierende Beitrag dieses Strukturmotivs abgeschätzt werden kann. Dieser Wert dient als Anhaltspunkt für die maximale Stabilität selbstständig faltender β-Haarnadeln und als Referenz zur spektroskopischen Abschätzung der Stabilität verwandter Peptide in Lösung.

Drug-induced stabilisation of a mismatched C-T base pair in a DNA hairpin

Co-reporter:Cathal T. Gallagher and Mark S. Searle

Chemical Communications 2003 (Issue 15) pp:1814-1815

Publication Date(Web):01 Jul 2003

DOI:10.1039/B305337G

We have shown that a key feature of drug binding, namely specific G-C base pair recognition at a 5′-TG step, can induce a number of novel structural features when an extrahelical base is inserted in close proximity to the drug binding site; we have clearly demonstrated the formation of a stabilised C-T mismatched base pair at a non-terminal site.

Electrospray ionisation mass spectrometric detection of weak non-covalent interactions in nogalamycin–DNA complexes

Co-reporter:Michelle L. Colgrave, Jennifer L. Beck, Margaret M. Sheil and Mark S. Searle

Chemical Communications 2002 (Issue 6) pp:556-557

Publication Date(Web):15 Feb 2002

DOI:10.1039/B200235N

We demonstrate the use of electrospray ionisation mass spectrometry (ESI-MS) in high salt solutions for the analysis of weak non-covalent complexes of the anthracycline antibiotic nogalamycin with novel DNA hairpin structures; high signal-to-noise ratios for the complexes in the absence of bound Na+ ions permits relative binding affinities to be estimated.

Structure of a Drug-Induced DNA T-Bulge: Implications for DNA Frameshift Mutations

Co-reporter:Michelle L. Colgrave Dr.;Huw E. L. Williams Dr. Dr.

Angewandte Chemie 2002 Volume 114(Issue 24) pp:

Publication Date(Web):12 DEC 2002

DOI:10.1002/ange.200290038

Eine ausgebeulte Konformation, in der die Base T3 aus der helicalen Packung der neuartigen Haarnadel-DNA H2 heraussteht, erlaubt die komplementäre Paarung downstream zur Intercalationsstelle (siehe Bild). Diese Konformation wird durch die Bindung von Nogalamycin stabilisiert, was durch detaillierte Analyse der chemischen Verschiebungen und NOE-Daten gezeigt wird.

Structure of a Drug-Induced DNA T-Bulge: Implications for DNA Frameshift Mutations

Co-reporter:Michelle L. Colgrave Dr.;Huw E. L. Williams Dr. Dr.

Angewandte Chemie International Edition 2002 Volume 41(Issue 24) pp:

Publication Date(Web):12 DEC 2002

DOI:10.1002/anie.200290039

A bulged conformation in which the T3 base is flipped-out of the helical stack of the novel DNA hairpin H2 allowing complementary pairing downstream of the intercalation site (see picture) is stabilized by binding of the drug nogalamycin. This finding was readily confirmed by detailed analysis of the chemical shift and NOE data.

Binding of anthracycline antibiotic nogalamycin to the site of a DNA single strand break engineered between two co-axially stacked hairpins

Co-reporter:Michelle L. Colgrave, Huw E. L. Williams and Mark S. Searle

Chemical Communications 2001 (Issue 4) pp:315-316

Publication Date(Web):31 Jan 2001

DOI:10.1039/B009904J

We show by NMR that the DNA sequence 5′-GCtext-decoration:underline"GAAGCACtext-decoration:underline"GAAGT folds to form an intramolecular double hairpin structure containing 5′-GAA loops with co-axial stacking of the two hairpins significantly increasing their stability; the anthracycline antibiotic nogalamycin is able to intercalate between the two hairpins (5′-TG step) despite the break in the phosphodiester backbone.

Design of histidine-Zn2+ binding sites within a β-hairpin peptide: enhancement of β-sheet stability through metal complexation

Co-reporter:Geoffrey Platt, Chun-Wa Chung and Mark S. Searle

Chemical Communications 2001 (Issue 13) pp:1162-1163

Publication Date(Web):08 Jun 2001

DOI:10.1039/B100702P

We describe the design and characterisation of two simple ‘metalloproteins’ based on Zn2+ co-ordination sites involving histidine residues close to the N- and C-termini of two β-hairpin peptides (His2-β, KHYTVSINGKKITVHI and His3-β, HKHYTV-SINGKKITVHI); we show by NMR and circular dichroism spectroscopy that Zn2+ complexation co-operatively enhances the stability of these partially pre-organised β-sheet peptides.

Recognition and Stabilization of Quadruplex DNA by a Potent New Telomerase Inhibitor: NMR Studies of the 2:1 Complex of a Pentacyclic Methylacridinium Cation with d(TTAGGGT)4

Co-reporter:Evripidis Gavathiotis;Robert A. Heald;Malcolm F. G. Stevens Dr.

Angewandte Chemie International Edition 2001 Volume 40(Issue 24) pp:

Publication Date(Web):18 DEC 2001

DOI:10.1002/1521-3773(20011217)40:24<4749::AID-ANIE4749>3.0.CO;2-I

Guided by 24 drug–DNA NOEs, the complex formed between RHPS4 (a novel fluorinated polycyclic methylacridinium salt with potent telomerase activity) and d(TTAGGGT)₄ has been modeled by using MD simulations with drug molecules intercalated at the ApG and GpT steps. A low-energy structure for the 2:1 complex has a partial positive charge on the acridine 13-N atom (which acts as a pseudo potassium ion) positioned above the center of the G-tetrad (see picture).

Recognition and Stabilization of Quadruplex DNA by a Potent New Telomerase Inhibitor: NMR Studies of the 2:1 Complex of a Pentacyclic Methylacridinium Cation with d(TTAGGGT)4

Co-reporter:Evripidis Gavathiotis;Robert A. Heald;Malcolm F. G. Stevens Dr.

Angewandte Chemie 2001 Volume 113(Issue 24) pp:

Publication Date(Web):18 DEC 2001

DOI:10.1002/1521-3757(20011217)113:24<4885::AID-ANGE4885>3.0.CO;2-3

Gestützt auf 24 Wirkstoff-DNA-NOEs: Ein Komplex aus einem neuen fluorierten polycyclischen Methylacridinium-Salz (RHPS4) mit hoher Telomerase-Aktivität und d(TTAGGGT)₄ wurde mithilfe von Moleküldynamiksimulationen unter Berücksichtigung intercalierter Wirkstoffmoleküle zwischen den ApG- und GpT-Stellen (siehe Abbildung) untersucht. In einer Niedrigenergiestruktur des 2:1-Komplexes trägt das Acridin-13-N-Atom, das hier als Pseudokaliumion fungiert und oberhalb des G-Tetrade-Zentrums angeordnet ist, eine positive Teilladung.

Do interstrand hydrogen bonds contribute to β-hairpin peptide stability in solution? IR analysis of peptide folding in water

Co-reporter:Christopher S. Colley, Samuel R. Griffiths-Jones, Michael W. George and Mark S. Searle

Chemical Communications 2000 (Issue 7) pp:593-594

Publication Date(Web):20 Mar 2000

DOI:10.1039/B000426J

The amide I carbonyl stretch in the IR spectrum, together with 1H NMR Hα chemical shifts, have been used to investigate the folding of a 16-residue β-hairpin peptide in water: while Hα shifts are consistent with a significant population of the folded state (ca. 40%), we see no features in the IR spectrum in the amide I region to suggest a significant contribution from interstrand hydrogen bonds, although at high peptide concentration (10 mM) the appearance of a new band at 1616 cm−1 is consistent with the onset of irreversible peptide aggregation.

Steady state and time-resolved IR spectroscopy of the native and unfolded states of bovine ubiquitin: protein stability and temperature-jump kinetic measurements of protein folding at low pH

Co-reporter:Christopher S. Colley, Ian P. Clark, Samuel R. Griffiths-Jones, Michael W. George and Mark S. Searle

Chemical Communications 2000 (Issue 16) pp:1493-1494

Publication Date(Web):19 Jul 2000

DOI:10.1039/B003768K

Infrared spectroscopy has been used to characterise the folded and unfolded states of bovine ubiquitin (a small protein of 76 residues) under acidic conditions (pH ∼1); fast time-resolved measurements of protein unfolding, initiated by a laser-induced temperature-jump of ∼8 °C, shows rapid refolding and β-sheet secondary structure formation on a timescale of a few milliseconds.

Sequential Barriers and an Obligatory Metastable Intermediate Define the Apparent Two-state Folding Pathway of the Ubiquitin-like PB1 Domain of NBR1

Co-reporter:Ping Chen, Jed Long, Mark S. Searle

Journal of Molecular Biology (7 March 2008) Volume 376(Issue 5) pp:1463-1477

Publication Date(Web):7 March 2008

DOI:10.1016/j.jmb.2007.12.052

The 90-residue N-terminal Phox and Bem1p (PB1) domain of NBR1 forms an α/β ubiquitin-like fold. Kinetic analysis using stopped-flow fluorescence reveals two-state kinetics; however, nonlinear effects in the denaturant dependence of the unfolding data demonstrate changes in the position of the rate-limiting barrier along the folding coordinate as the folding conditions change. The kinetics of wt-PB1 and several mutants show that this curvature is consistent with a single-pathway mechanism involving sequential transition states (TS1 and TS2) separated by a transiently populated high-energy intermediate, rather than movement of the transition state on a broad energy plateau. We show that the two transition states within the sequential model represent structurally and thermodynamically distinct species. TS1 is a collapsed state (αTS1 = 0.71) with a large enthalpic barrier to formation that is rate-limiting under conditions that strongly favour folding. TS2 is highly native-like (αTS2 = 0.93) and represents a late entropic barrier to formation of the native state. In support of the sequential transition state mechanism, we show that the G62A helix 2 substitution stabilises TS1 and the intermediate to such an extent that the latter becomes significantly populated, leading to the observation of a fast kinetic phase representing the initial U → I transition, with TS2 (αTS2 = 0.87) becoming rate-limiting. The folding rate is not retarded by populating an intermediate, which would be expected for a misfold state, but is accelerated, suggesting that the I state is productive and on-pathway. The results show that the apparent two-state folding of the wt-PB1 domain occurs along a well-defined pathway involving structurally and thermodynamically distinct sequential transition states and an obligatory metastable intermediate that represents a productive local minimum in the energy landscape that increases the efficiency of barrier crossing through favourable effects on the entropy of activation.

Folding Topology of a Bimolecular DNA Quadruplex Containing a Stable Mini-hairpin Motif within the Diagonal Loop

Co-reporter:Graham D. Balkwill, Thomas P. Garner, Huw E.L. Williams, Mark S. Searle

Journal of Molecular Biology (6 February 2009) Volume 385(Issue 5) pp:1600-1615

Publication Date(Web):6 February 2009

DOI:10.1016/j.jmb.2008.11.050

We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G3ACGTAGTG3)2 containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G3T4G3)2 dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex–duplex interface that are not present in the structure with the T4-loop sequence. A truncated version of the bimolecular quadruplex d(G2ACGTAGTG2)2, with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.

Bile Acid Interactions with Rabbit Ileal Lipid Binding Protein and an Engineered Helixless Variant Reveal Novel Ligand Binding Properties of a Versatile β-Clam Shell Protein Scaffold

Co-reporter:Nikolaos Kouvatsos, Victoria Thurston, Kathryn Ball, Neil J. Oldham, ... Mark S. Searle

Journal of Molecular Biology (31 August 2007) Volume 371(Issue 5) pp:1365-1377

Publication Date(Web):31 August 2007

DOI:10.1016/j.jmb.2007.06.024

The intracellular ileal lipid binding proteins (ILBPs) are involved in the transport and enterohepatic circulation of bile acids. ILBPs from different species show high sequence and structural homology and have been shown to bind multiple bile acid ligands with differing degrees of selectivity and positive co-operativity. Human ILBP binds bile acid derivatives in a well-characterised 2:1 ligand:protein complex, however, we show that the highly homologous rabbit ILBP (82% sequence identity) with seven conservative substitutions preferentially binds multiple conjugated deoxycholate ligands in a novel 3:1 binding mode essentially within the same β-clam shell structure. We have extended these studies to investigate the role of the α-helical capping motif (residues 9–35) in controlling the dimensions of the binding cavity and ligand uptake. Substituting the α-helical motif (residues 9–35) with a short Gly-Gly-Ser-Gly linker dramatically affects the protein stability such that under physiological conditions the mutant (Δα–ILBP) is highly disordered. However, we show that the inability of the mutant to adopt a stable three-dimensional structure under these conditions is no barrier to binding ligands with near-native affinity. These structural modifications not only demonstrate the possibility of strong coupling between ligand binding and protein folding, but result in changes in bile acid selectivity and binding stoichiometry, which we characterise in detail using isothermal calorimetry and mass spectrometry.

Structural Rearrangement in an RsmA/CsrA Ortholog of Pseudomonas aeruginosa Creates a Dimeric RNA-Binding Protein, RsmN

Co-reporter:Elizabeth R. Morris, Gareth Hall, Chan Li, Stephan Heeb, ... Mark S. Searle

Structure (3 September 2013) Volume 21(Issue 9) pp:1659-1671

Publication Date(Web):3 September 2013

DOI:10.1016/j.str.2013.07.007

•Phenotypic complementation identifies a CsrA/RsmA family member from P. aeruginosa•Crystallography reveals a dimeric fold for RsmN•The RsmN complex was solved with a hairpin motif from the noncoding sRNA RsmZ-2•Details of binding affinity and specificity with target RNA 5′-ANGGAN motifs are revealedIn bacteria, the highly conserved RsmA/CsrA family of RNA-binding proteins functions as global posttranscriptional regulators acting on mRNA translation and stability. Through phenotypic complementation of an rsmA mutant in Pseudomonas aeruginosa, we discovered a family member, termed RsmN. Elucidation of the RsmN crystal structure and that of the complex with a hairpin from the sRNA, RsmZ, reveals a uniquely inserted α helix, which redirects the polypeptide chain to form a distinctly different protein fold to the domain-swapped dimeric structure of RsmA homologs. The overall β sheet structure required for RNA recognition is, however, preserved with compensatory sequence and structure differences, allowing the RsmN dimer to target binding motifs in both structured hairpin loops and flexible disordered RNAs. Phylogenetic analysis indicates that, although RsmN appears unique to P. aeruginosa, homologous proteins with the inserted α helix are more widespread and arose as a consequence of a gene duplication event.

Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling

Co-reporter:Jed Long, Thomas P. Garner, Maya J. Pandya, C. Jeremy Craven, ... Mark S. Searle

Journal of Molecular Biology (12 February 2010) Volume 396(Issue 1) pp:178-194

Publication Date(Web):12 February 2010

DOI:10.1016/j.jmb.2009.11.032

The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-κB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Paget's disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-κB signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (Kdim ∼ 4−12 μM at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-κB luciferase reporter assays, are associated with reduced NF-κB activity compared with wt-p62.

Structure and folding dynamics of a DNA hairpin with a stabilising d(GNA) trinucleotide loop: influence of base pair mis-matches and point mutations on conformational equilibria

Co-reporter:Graham D. Balkwill, Huw E. L. Williams and Mark S. Searle

Organic & Biomolecular Chemistry 2007 - vol. 5(Issue 5) pp:NaN839-839

Publication Date(Web):2007/01/25

DOI:10.1039/B616820E

Hairpins are known to play specific roles in DNA– and RNA–protein recognition. Various disease states are thought to originate from the ill-timed formation of a hairpin loop during transcription, particularly in the context of triplet repeats which are associated with myotonic dystrophy, fragile X syndrome and other genetic disorders. An understanding of nucleic acid folding mechanisms requires a detailed appreciation of the timescales of these local folding events, a characterisation of the conformational equilibria that exist in solution and the influence of point mutations on the relative stabilities of the different species. We investigate using NMR and CD spectroscopy the structure and dynamics of a DNA hairpin containing a highly stabilising cGNAg loop. The single-stranded 13-mer 5′-d(GCTACGNAGTCGC) with N = T folds to form a hairpin structure which accommodates a C–T mis-matched base pair within the double-stranded stem region. The hairpin is in equilibrium with a double-stranded duplex form with the mixture of two interconverting conformations in slow exchange on the NMR timescale (1–2 s–1 at 308 K). We are able to characterise the dynamics of the interconversion process by NMR magnetisation transfer and by CD stopped-flow kinetic experiments. The latter shows that the hairpin folds too rapidly to detect by this method (>500 s–1) and forms in a “kinetic overshoot” followed by a much slower equilibration to a mixture of conformations (∼0.13 s–1 at 298 K). A point mutation that converts the GTA to a GAA loop sequence destabilises the intermolecular duplex structure and enables us to unambiguously assign the various dynamic processes that are taking place.

Selectivity of small molecule ligands for parallel and anti-parallel DNA G-quadruplex structures

Co-reporter:Thomas P. Garner, Huw E. L. Williams, Katarzyna I. Gluszyk, Stephen Roe, Neil J. Oldham, Malcolm F. G. Stevens, John E. Moses and Mark S. Searle

Organic & Biomolecular Chemistry 2009 - vol. 7(Issue 20) pp:NaN4200-4200

Publication Date(Web):2009/08/14

DOI:10.1039/B910505K

We report CD, ESI-MS and molecular modelling studies of ligand binding interactions with DNA quadruplex structures derived from the human telomeric repeat sequence (h-Tel) and the proto-oncogenic c-kit promoter sequence. These sequences form anti-parallel (both 2 + 2 and 3 + 1) and parallel conformations, respectively, and demonstrate distinctively different degrees of structural plasticity in binding ligands. With h-Tel, we show that an extended heteroaromatic 1,4-triazole (TRZ), designed to exploit π-stacking interactions and groove-specific contacts, shows some selectivity for parallel folds, however, the polycyclic fluorinated acridinium cation (RHPS4), which is a similarly potent telomerase inhibitor, shows selectivity for anti-parallel conformations implicating favourable interactions with lateral and diagonal loops. In contrast, the unique c-kit parallel-stranded quadruplex shows none of the structural plasticity of h-Tel with either ligand. We show by quantitative ESI-MS analysis that both sequences are able to bind a ligand on either end of the quadruplex. In the case of h-Tel the two sites have similar affinities, however, in the case of the c-kit quadruplex the affinities of the two sites are different and ligand-dependent. We demonstrate that two different small molecule architectures result in significant differences in selectivity for parallel and anti-parallel quadruplex structures that may guide quadruplex targeted drug-design.

Ligand selectivity in stabilising tandem parallel folded G-quadruplex motifs in human telomeric DNA sequences

Co-reporter:Alex R. O. Cousins, Dougal Ritson, Pallavi Sharma, Malcolm F. G. Stevens, John E. Moses and Mark S. Searle

Chemical Communications 2014 - vol. 50(Issue 96) pp:NaN15205-15205

Publication Date(Web):2014/10/23

DOI:10.1039/C4CC07487D

Biophysical studies of ligand interactions with three human telomeric repeat sequences (d(AGGG(TTAGGG)n, n = 3, 7 and 11)) show that an oxazole-based ‘click’ ligand, which induces parallel folded quadruplexes, preferentially stabilises longer telomeric repeats providing evidence for selectivity in binding at the interface between tandem quadruplex motifs.