A central challenge in structure-based ligand design is the accurate prediction of binding free energies. Here we apply alchemical free energy calculations in explicit solvent to predict ligand binding in a model cavity in T4 lysozyme. Even in this simple site, there are challenges. We made systematic improvements, beginning with single poses from docking, then including multiple poses, additional protein conformational changes, and using an improved charge model. Computed absolute binding free energies had an RMS error of 1.9 kcal/mol relative to previously determined experimental values. In blind prospective tests, the methods correctly discriminated between several true ligands and decoys in a set of putative binders identified by docking. In these prospective tests, the RMS error in predicted binding free energies relative to those subsequently determined experimentally was only 0.6 kcal/mol. X-ray crystal structures of the new ligands bound in the cavity corresponded closely to predictions from the free energy calculations, but sometimes differed from those predicted by docking. Finally, we examined the impact of holding the protein rigid, as in docking, with a view to learning how approximations made in docking affect accuracy and how they may be improved.

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 ␤-aminoketones, which exhibited IC 50 values of ϳ2 M. 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 ϳ0.6 M 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. Thyroid hormone receptors (TRs)3 regulate development, growth, and metabolism (1, 2). The TRs are nuclear receptors (NR), part of a superfamily whose members function as hormone-activated transcription factors (3). The majority of thyroid hormone responses are induced by regulation of transcription by the thyroid hormone T3 (4). Two genes, THRA and THRB encode the two protein isoforms TR␣ and TR␤, which yield four distinct subtypes by alternative splicing (5). Several functional domains of TRs have been identified: a ligand-independent transactivation domain (AF-1) on the amino terminus, a central DNA binding domain, a ligand binding domain (LBD), and a carboxylterminal ligand dependent activation function (AF-2) (6). TR binds specific sequences of DNA in the 5Ј-flanking regions of T3-responsive genes, known as thyroid response elements, most often as a heterodimer with the retinoid X receptor (7). Both unliganded and liganded TRs can bind thyroid response elements and regulate genes under their control. The unliganded TR complex can recruit a nuclear receptor corepressor (NCoR) or a silencing mediator of retinoic acid to silence basal transcription (8). In the presence of T3, TRs undergo a conformational change with the result that the composition of the coregulator complex can change with strong effects on transcriptional regulation. Several coactivator proteins have been identified (9). The best studied group of coactivators is the p160 or steroid receptor coactivator (SRC) proteins (7) including SRC1 (10), SRC2 (11,12), and SRC3 (13). Another group of ligand-dependent-interactin...

Pre-organization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein, and consequently enzymes can be dramatically stabilized by substitutions that attenuate the size and pre-organization “strain” of the active site. How this stability-activity trade-off constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities, or how these new activities manifest structurally. These questions are both germane and easy to study in β-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of β-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C β-lactamases which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100- to 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability – from 1.7 to 4.1 kcal/mol – consistent with the function-stability hypothesis. Intriguingly, several of the substitutions were 10 – 20 Å from the catalytic serine; the question arose how they conferred extended-spectrum activity. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by x-ray crystallography. Distinct mechanisms of action are revealed for each mutant, including changes in the flexibility and ground state structures of the enzyme. These results explain the structural bases for the antibiotic resistance conferred by these substitutions, and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.

The thyroid hormone receptor regulates a diverse set of genes that control processes from embryonic development to adult homeostasis. Upon binding of thyroid hormone, the thyroid receptor releases corepressor proteins and undergoes a conformational change that allows for the interaction of coactivating proteins necessary for gene transcription. This interaction is mediated by a conserved motif, termed the NR box, found in many coregulators. Recent work has demonstrated that differentially assembled coregulator complexes can elicit specific biological responses. However, the mechanism for the selective assembly of these coregulator complexes has yet to be elucidated. To further understand the principles underlying thyroid receptor-coregulator selectivity, we designed a high-throughput in vitro binding assay to measure the equilibrium affinity of thyroid receptor to a library of potential coregulators in the presence of different ligands including the endogenous thyroid hormone T3, synthetic thyroid receptor ␤-selective agonist GC-1, and antagonist NH-3. Using this homogenous method several coregulator NR boxes capable of associating with thyroid receptor at physiologically relevant concentrations were identified including ones found in traditional coactivating proteins such as SRC1, SRC2, TRAP220, TRBP, p300, and ARA70; and those in coregulators known to repress gene activation including RIP140 and DAX-1. In addition, it was discovered that the thyroid receptor-coregulator binding patterns vary with ligand and that this differential binding can be used to predict biological responses. Finally, it is demonstrated that this is a general method that can be applied to other nuclear receptors and can be used to establish rules for nuclear receptorcoregulator selectivity.

Lys67 is essential for the hydrolysis reaction mediated by class C b-lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C b-lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2-to 3-fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C b-lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61-fold decrease in k cat , the deacylation rate. We have determined the structure of the K67R mutant by X-ray crystallography both in apo and transition state-analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C b-lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.