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Biomedical subjects

Robert J Fletterick

Publications and source records attributed to Robert J Fletterick.

At least 19 recordsLinked to original sources
PubMed2006

Structural basis for unique mechanisms of folding and hemoglobin binding by a malarial protease.

Falcipain-2 (FP2), the major cysteine protease of the human malaria parasite Plasmodium falciparum, is a hemoglobinase and promising drug target. Here we report the crystal structure of FP2 in complex with a protease inhibitor, cystatin. The FP2 structure reveals two previously undescribed cysteine protease structural motifs, designated FP2(nose) and FP2(arm), in addition to details of the active site that will help focus inhibitor design. Unlike most cysteine proteases, FP2 does not require a prodomain but only the short FP2(nose) motif to correctly fold and gain catalytic activity. Our structure and mutagenesis data suggest a molecular basis for this unique mechanism by highlighting the functional role of two Tyr within FP2(nose) and a conserved Glu outside this motif. The FP2(arm) motif is required for hemoglobinase activity. The structure reveals topographic features and a negative charge cluster surrounding FP2(arm) that suggest it may serve as an exo-site for hemoglobin binding. Motifs similar to FP2(nose) and FP2(arm) are found only in related plasmodial proteases, suggesting that they confer malaria-specific functions.

Amino Acid Sequence↗
PubMed2006

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.

Amino Acid Sequence↗
PubMed2006

A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding.

To provide alternative methods for regulation of gene transcription initiated by the binding of thyroid hormone (T3) to the thyroid receptor (TR), we have developed a high-throughput method for discovering inhibitors of the interaction of TR with its transcriptional coactivators. The screening method is based on fluorescence polarization (FP), one of the most sensitive and robust high-throughput methods for the study of protein-protein interactions. A fluorescently labeled coactivator is excited by polarized light. The emitted polarized light is a function of the molecular properties of the labeled coactivator, especially Brownian molecular rotation, which is very sensitive to changes in the molecular mass of the labeled complex. Dissociation of hormone receptor from fluorescently labeled coactivator peptide in the presence of small molecules can be detected by this competition method, and the assay can be performed in a high-throughput screening format. Hit compounds identified by this method are evaluated by several secondary assay methods, including a dose-response analysis, a semiquantitative glutathione-S-transferase assay, and a hormone displacement assay. Subsequent in vitro transcription assays can detect inhibition of thyroid signaling at low micromolar concentrations of small molecules in the presence of T3.

Acetyltransferases↗
PubMed2006

An atomic model of the thin filament in the relaxed and Ca2+-activated states.

Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the "core domain" of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.

Actins↗
PubMed2006

Dynamics of the C-terminal region of TnI in the troponin complex in solution.

The determination of crystal structures of the troponin complex (Takeda et al. 2003. Nature. 424:35-41; Vinogradova et al. 2005. Proc. Natl. Acad. Sci. USA. 102:5038-5043) has advanced knowledge of the regulation of muscle contraction at the molecular level. However, there are domains important for actin binding that are not visualized. We present evidence that the C-terminal region of troponin I (TnI residues 135-182) is flexible in solution and has no stable secondary structure. We use NMR spectroscopy to observe the backbone dynamics of skeletal [2H, 13C, 15N]-TnI in the troponin complex in the presence of Ca2+ or EGTA/Mg2+. Residues in this region give stronger signals than the remainder of TnI, and chemical shift index values indicate little secondary structure, suggesting a very flexible region. This is confirmed by NMR relaxation measurements. Unlike TnC and other regions of TnI in the complex, the C-terminal region of TnI is not affected by Ca2+ binding. Relaxation measurements and reduced spectral density analysis are consistent with the C-terminal region of TnI being a tethered domain connected to the rest of the troponin complex by a flexible linker, residues 137-146, followed by a collapsed region with at most nascent secondary structure.

Actins↗
PubMed2005

Discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators.

Thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic beta-aminoketones, which exhibited IC50 values of approximately 2 microm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of approximately 0.6 microm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.

Binding, Competitive↗
PubMed2005

Calcium-dependent changes in the flexibility of the regulatory domain of troponin C in the troponin complex.

With the recent advances in structure determination of the troponin complex, it becomes even more important to understand the dynamics of its components and how they are affected by the presence or absence of Ca(2+). We used NMR techniques to study the backbone dynamics of skeletal troponin C (TnC) in the complex. Transverse relaxation-optimized spectroscopy pulse sequences and deuteration of TnC were essential to assign most of the TnC residues in the complex. Backbone amide (15)N relaxation times were measured in the presence of Ca(2+) or EGTA/Mg(2+). T(1) relaxation times could not be interpreted precisely, because for a molecule of this size, the longitudinal backbone amide (15)N relaxation rate due to chemical shift anisotropy and dipole-dipole interactions becomes too small, and other relaxation mechanisms become relevant. T(2) relaxation times were of the expected magnitude for a complex of this size, and most of the variation of T(2) times in the presence of Ca(2+) could be explained by the anisotropy of the complex, suggesting a relatively rigid molecule. The only exception was EF-hand site III and helix F immediately after, which are more flexible than the rest of the molecule. In the presence of EGTA/Mg(2+), relaxation times for residues in the C-domain of TnC are very similar to values in the presence of Ca(2+), whereas the N-domain becomes more flexible. Taken together with the high flexibility of the linker between the two domains, we concluded that in the absence of Ca(2+), the N-domain of TnC moves independently from the rest of the complex.

Animals↗
PubMed2005

Ca(2+)-regulated structural changes in troponin.

Troponin senses Ca2+ to regulate contraction in striated muscle. Structures of skeletal muscle troponin composed of TnC (the sensor), TnI (the regulator), and TnT (the link to the muscle thin filament) have been determined. The structure of troponin in the Ca(2+)-activated state features a nearly twofold symmetrical assembly of TnI and TnT subunits penetrated asymmetrically by the dumbbell-shaped TnC subunit. Ca ions are thought to regulate contraction by controlling the presentation to and withdrawal of the TnI inhibitory segment from the thin filament. Here, we show that the rigid central helix of the sensor binds the inhibitory segment of TnI in the Ca(2+)-activated state. Comparison of crystal structures of troponin in the Ca(2+)-activated state at 3.0 angstroms resolution and in the Ca(2+)-free state at 7.0 angstroms resolution shows that the long framework helices of TnI and TnT, presumed to be a Ca(2+)-independent structural domain of troponin are unchanged. Loss of Ca ions causes the rigid central helix of the sensor to collapse and to release the inhibitory segment of TnI. The inhibitory segment of TnI changes conformation from an extended loop in the presence of Ca2+ to a short alpha-helix in its absence. We also show that Anapoe, a detergent molecule, increases the contractile force of muscle fibers and binds specifically, together with the TnI switch helix, in a hydrophobic pocket of TnC upon activation by Ca ions.

Animals↗
PubMed2005

Conformational adaptation of nuclear receptor ligand binding domains to agonists: potential for novel approaches to ligand design.

Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5' extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5' iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRbeta structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3' benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.

Acetates↗
PubMed2005

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1.

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Animals↗
PubMed2005

Molecular modelling of the androgen receptor axis: rational basis for androgen receptor intervention in androgen-independent prostate cancer.

Androgen depletion in combination with antiandrogenic agents is initially highly effective for treating prostate cancer, and is the recommended treatment for more advanced or higher-grade tumours. However, many tumours eventually become insensitive to androgens, even though the androgen receptor (AR) continues to be expressed. Computational chemistry combined with structural analysis of nuclear receptors and determination of binding affinities of natural and designed coregulators (coactivators and corepressors) provides the theoretical framework for the rational design of novel therapeutic agents directed at the AR. Adding alternative groups to various sites throughout the receptor can alter the conformation of the molecule and its functional binding with coactivators or corepressors. Possible molecules can be identified thoroughly and systematically using intelligent high-throughput screening and FASTrack chemistry (three-dimensional crystallography). Applying these techniques should eventually result in therapeutic agents for androgen-independent prostate cancer that can block binding of AR coactivators while simultaneously increasing binding of AR corepressors.

Androgen Antagonists↗
PubMed2004

The molecular mechanisms of coactivator utilization in ligand-dependent transactivation by the androgen receptor.

Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism. Here, we report that the AR-LBD interacts with both FXXLF motifs and a subset of LXXLL motifs and that contacts with these LXXLL motifs are both necessary and sufficient for SRC-mediated AR regulation of transcription. Crystal structures of the activated AR in complex with both recruitment motifs reveal that side chains unique to the AR-LBD rearrange to bind either the bulky FXXLF motifs or the more compact LXXLL motifs and that AR utilizes subsidiary contacts with LXXLL flanking sequences to discriminate between LXXLL motifs.

Amino Acid Motifs↗
PubMed2004

Thyroxine-thyroid hormone receptor interactions.

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3), 3,5,3'-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T(4), 3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless, T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH, our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design.

Animals↗
PubMed2004

Recognition and accommodation at the androgen receptor coactivator binding interface.

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.

Amino Acid Motifs↗
PubMed2004

Characterization of structural determinants of granzyme B reveals potent mediators of extended substrate specificity.

Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile99, Tyr174, Arg192, and Asn218. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile99 completely defines and predicts P2 specificity, primarily through the binding constant Km. Asn218 variants have minor effects alone but in combination with mutations at Arg192 and Ile99 alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn218 variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn218 not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members.

Alanine↗
PubMed2004

Crystal structure of kinesin regulated by Ca(2+)-calmodulin.

Kinesins orchestrate cell division by controlling placement of chromosomes. Kinesins must be precisely regulated or else cell division fails. Calcium, a universal second messenger in eukaryotes, and calmodulin regulate some kinesins by causing the motor to dissociate from its biological track, the microtubule. Our focus was the mechanism of calcium regulation of kinesin at atomic resolution. Here we report the crystal structure of kinesin-like calmodulin-binding protein (KCBP) from potato, which was resolved to 2.3 A. The structure reveals three subdomains of the regulatory machinery located at the C terminus extension of the kinesin motor. Calmodulin that is activated by Ca2+ ions binds to an alpha-helix positioned on the microtubule-binding face of kinesin. A negatively charged segment following this helix competes with microtubules. A mimic of the conventional kinesin neck, connecting the calmodulin-binding helix to the KCBP motor core, links the regulatory machine to the kinesin catalytic cycle. Together with biochemical data, the crystal structure suggests that Ca(2+)-calmodulin inhibits the binding of KCBP to microtubules by blocking the microtubule-binding sites on KCBP.

Arabidopsis Proteins↗
PubMed2003

Ligand selectivity by seeking hydrophobicity in thyroid hormone receptor.

Selective therapeutics for nuclear receptors would revolutionize treatment for endocrine disease. Specific control of nuclear receptor activity is challenging because the internal cavities that bind hormones can be virtually identical. Only one highly selective hormone analog is known for the thyroid receptor, GC-24, an agonist for human thyroid hormone receptor beta. The compound differs from natural hormone in benzyl, substituting for an iodine atom in the 3' position. The benzyl is too large to fit into the enclosed pocket of the receptor. The crystal structure of human thyroid hormone receptor beta at 2.8-A resolution with GC-24 bound explains its agonist activity and unique isoform specificity. The benzyl of GC-24 is accommodated through shifts of 3-4 A in two helices. These helices are required for binding hormone and positioning the critical helix 12 at the C terminus. Despite these changes, the complex associates with coactivator as tightly as human thyroid hormone receptor bound to thyroid hormone and is fully active. Our data suggest that increased specificity of ligand recognition derives from creating a new hydrophobic cluster with ligand and protein components.

Acetates↗