Discovery Of Binding Sites Research Articles

Experimental approaches to investigate biophysical interactions between homeodomain transcription factors and DNA.

Homeodomain transcription factors (TFs) bind to specific DNA sequences to regulate the expression of target genes. Structural work has provided insight into molecular identities and aided in unraveling structural features of these TFs. However, the detailed affinity and specificity by which these TFs bind to DNA sequences is still largely unknown. Qualitative methods, such as DNA footprinting, Electrophoretic Mobility Shift Assays (EMSAs), Systematic Evolution of Ligands by Exponential Enrichment (SELEX), Bacterial One Hybrid (B1H) systems, Surface Plasmon Resonance (SPR), and Protein Binding Microarrays (PBMs) have been widely used to investigate the biochemical characteristics of TF-DNA binding events. In addition to these qualitative methods, bioinformatic approaches have also assisted in TF binding site discovery. Here we discuss the advantages and limitations of these different approaches, as well as the benefits of utilizing more quantitative approaches, such as Mechanically Induced Trapping of Molecular Interactions (MITOMI), Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC), in determining the biophysical basis of binding specificity of TF-DNA complexes and improving upon existing computational approaches aimed at affinity predictions.

Multilayer Annotation Strategy AnnoSePS: Disentangling the Intricate Structure of Selenium-Containing Polysaccharides Based on Preferential Fragmentation Patterns.

Precision mapping of selenium at structural and position levels poses significant challenges in selenium-containing polysaccharide identification. Due to the absence of reference spectra, database-centric approaches are still limited in the discovery of selenium binding sites and distinction among different isomeric structures. A multilayer annotation strategy, AnnoSePS, is proposed for achieving the identification of seleno-substituent and the unbiased profiling of polysaccharides. Applying Snoop-triggered multiple reaction monitoring (Snoop-MRM) identified multidimensional monosaccharides in selenium-containing polysaccharides. Galactose, galacturonic acid, and glucose were the predominant monosaccharides with a molar ratio of 25.19, 19.45, and 11.72, respectively. Selenium present in seleno-rhamnose was found to substitute the hydroxyl group located at C-1 positions through the formation of a Se-H bond. Ions C6H9O3Se-, C6H7O3Se-, C5H5O3Se-, C4H5O2Se-, C3H5O2Se-, C2H3O2Se-, and CHOSe- were defined as the characteristic fragments of seleno-rhamnose. The agglomerative hierarchical clustering algorithm is applied to group spectra from each run based on the characteristic information. Preferential fragmentation patterns in mass spectrometry are revealed by training a probabilistic model. A list of candidate oligosaccharides is generated by step-by-step browsing through the transition pairs for all reference spectra and applying the transitions (addition, insertion, removal, and substitution) to reference structures. Combining time course analyses revealed the linkage composition of selenium-containing oligosaccharides. Glycosidic linkages were annotated based on a synthesis-driven approach. T-Galactose (16.67 ± 5.23%) and T-Galacturonic acid (11.54 ± 4.66%) were the predominant linkage residues. As the database-independent mapping strategy, AnnoSePS makes it possible to comprehensively interrogate spectral data and dissect the fine structure of selenium-containing polysaccharides.

Known and potential benefits of applying herbicides with glutathione S-transferase inhibitors and inducers – a review

Abstract Weed resistance to herbicides has increased exponentially during the past 30 to 40 years, consequently reducing the number of effective products available to control certain species and populations. Future efforts should target not only the discovery of new protein binding sites and the development of new molecules, but also the revival of old molecules with reduced efficacy due to widespread herbicide resistance. The addition of herbicide synergists that inhibit metabolic pathways or enhance intrinsic plant stress is a possible solution to ameliorate the negative effects caused by the lack of new herbicide chemistries. Glutathione S-transferase (GST) enzymes are involved with numerous herbicide detoxification reactions and plant stress responses. This review approaches the potential use of natural and synthetic GST-inhibitors to enhance herbicidal activity or induce crop safety to provide effective, sustainable weed management strategies in the future.

Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM

Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside from canonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ion channels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution of these regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLIC by integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipids in the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipids were associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutant simulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, while a putative intersubunit site was preferred in open-state simulations. This work offers molecular details of GLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependent binding, and a multidisciplinary strategy for modeling protein-lipid interactions.

Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM.

Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside from canonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ion channels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution of these regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLIC by integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipids in the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipids were associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutant simulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, while a putative intersubunit site was preferred in open-state simulations. This work offers molecular details of GLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependent binding, and a multidisciplinary strategy for modeling protein-lipid interactions.

Abstract B053: Applying a new drug discovery platform for the discovery and development of inhibitors of DDR proteins APE1 and Pol eta

Abstract Quickly generating reliable hits from library screening and rapidly converting them into lead compounds, which can be verified in functional assays, is critical in early preclinical drug discovery. The expedited validation of “druggability” of targets and identification of modulators to advance to preclinical studies can significantly increase the success of drug discovery and development. Our SaXPy (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows (i) hit generation; (ii) mapping of protein-ligand interactions; (iii) assessment of target ligandability; (iv) discovery of novel and potential allosteric binding sites; and (v) hit-to-lead progression. These advances inform chemical tractability, downstream biology, and intellectual property. We describe here the application of SaXPy in the discovery and development of DNA Damage Response (DDR) inhibitors against DNA polymerase eta (Pol 𝝶 or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our studies resulted in the first crystal structures of these targets bound to small molecules and the discovery of novel, multiple binding sites, which we could quickly progress from hit-to-lead development and demonstrate functional activity in bioassays. Citation Format: Debanu Das, Matthew Duncton, Ashley Deacon, David Wilson III. Applying a new drug discovery platform for the discovery and development of inhibitors of DDR proteins APE1 and Pol eta [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B053.

Discovery and Exploration of Protein Kinase CK2 Binding Sites Using CK2α′Cys336Ser as an Exquisite Crystallographic Tool

The structural knowledge about protein kinase CK2 is dominated by crystal structures of human CK2α, the catalytic subunit of human CK2, and the product of the CSNK2A1 gene. In contrast, far fewer structures of CK2α′, its paralogous isoform and the product of the CSNK2A2 gene, have been published. However, according to a PDB survey, CK2α′ is the superior alternative for crystallographic studies because of the inherent potential of the single mutant CK2α′Cys336Ser to provide crystal structures with atomic resolution. In particular, a triclinic crystal form of CK2α′Cys336Ser is a robust tool to determine high-quality enzyme-ligand complex structures via soaking. In this work, further high-resolution CK2α′Cys336Ser structures in complex with selected ligands emphasizing this trend are described. In one of these structures, the “N-terminal segment site”, a small-molecule binding region never found in any eukaryotic protein kinase and holding the potential for the development of highly selective substrate-competitive CK2 inhibitors, was discovered. In order to also address the binding site for the non-catalytic subunit CK2β, which is inaccessible in these triclinic CK2α′Cys336Ser crystals for small molecules, a reliable path to a promising monoclinic crystal form of CK2α′Cys336Ser is presented. In summary, the quality of CK2α′Cys336Ser as an exquisite crystallographic tool is solidified.

A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows for (i) hit generation; (ii) the mapping of protein–ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq.

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false positive rates. Here, we present the R/Bioconductor package DEWSeq that makes use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing. The improvement not only relates to the number of binding sites (3.1-fold with known motifs for RBFOX2), but also their subcellular localization (1.9-fold of mitochondrial genes for FASTKD2) and structural targets (2.2-fold increase of stem-loop regions for SLBP. On several orthogonal CLIP-seq datasets, DEWSeq recovers a larger number of motif-containing binding sites (3.3-fold). DEWSeq is a well-documented R/Bioconductor package, scalable to adequate numbers of replicates, and tends to substantially increase the proportion and total number of RBP binding sites containing biologically relevant features.

FLARE: a fast and flexible workflow for identifying RNA editing foci

BackgroundFusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors.ResultsWe present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing.ConclusionsFLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available at https://github.com/YeoLab/FLARE.

Intermediate-aided allostery mechanism for α-glucosidase by Xanthene-11v as an inhibitor using residue interaction network analysis

Exploring allosteric inhibition and the discovery of new inhibitor binding sites are important studies in protein regulation mechanisms and drug discovery. Structural and network-based analyses of trajectories resulting from molecular dynamics (MD) simulations have been developed to discover protein dynamics, landscape, functions, and allosteric regions. Here, an experimentally suggested non-competitive inhibitor, xanthene-11v, was considered to explore its allosteric inhibition mechanism in α-glucosidase MAL12. Comparative structural and network analyses were applied to eight 250 ns independent MD simulations, four of which were performed in the free state and four of which were performed in ligand-bound forms. Projected two-dimensional free energy landscapes (FEL) were constructed from the probabilistic distribution of conformations along the first two principal components. The post-simulation analyses of the coordinates, side-chain torsion angles, non-covalent interaction networks, network communities, and their centralities were performed on α-glucosidase conformations and the intermediate sub-states. Important communities of residues have been found that connect the allosteric site to the active site. Some of these residues like Thr307, Arg312, TYR344, ILE345, Phe357, Asp406, Val407, Asp408, and Leu436 are the key messengers in the transition pathway between allosteric and active sites. Evaluating the probability distribution of distances between gate residues including Val407 in one community and Phe158, and Pro65 in another community depicted the closure of this gate due to the inhibitor binding. Six macro states of protein were deduced from the topology of FEL and analysis of conformational preference of free and ligand-bound systems to these macro states shows a combination of lock-and-key, conformational selection, and induced fit mechanisms are effective in ligand binding. All these results reveal structural states, allosteric mechanisms, and key players in the inhibition pathway of α-glucosidase by xanthene-11v.

Discovery of lipid binding sites in the ligand-gated ion channel GLIC through simulations and Cryo-EM.

Ligand-gated ion channels play an important role in electrochemical signal transduction in neurons and other excitable cells. Aside from canonical ligands, phospholipids have been shown to bind specifically and sometimes modulate several types of channels, but the structural details of such protein-lipid interactions remain unclear. Here, we present multiple lipid interaction sites in the pentameric ligand-gated ion channel, GLIC, by integrating cryo-electron microscopy and large-scale atomistic molecular dynamics simulations together with Markov state modeling. We identified 25 lipids in the closed state of GLIC - a conformation in which, to our knowledge, lipids have not been previously resolved. Both simulations and cryo-EM data predicted, for each subunit, three lipids in the inner leaflet and two in the outer leaflet. Further, we used simulations to make predictions about lipid interactions in the open state, resulting in six sites - three in each leaflet. Interactions of particular interest include one slow-exchanging interaction with a deeply buried lipid tail in the intersubunit space, where tail saturation levels played a role in the binding pose, and where the interaction could be broken due to a single mutation. An additional lipid interaction was preferential to the open state, potentially playing a role in stabilization of this state. This work provides molecular details of GLIC-lipid contacts, especially in the understudied closed state; a testable hypotheses for state-dependent binding; and a multidisciplinary strategy for modeling protein-lipid interactions.

Metabotropic glutamate receptor 1 alpha: a unique receptor variant with variable implications for Alzheimer's disease pathogenesis.

Metabotropic glutamate receptor 1 alpha: a unique receptor variant with variable implications for Alzheimer's disease pathogenesis.

Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP

Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.

Development of an Allostery Responsive Chromatographic Method for Screening Potential Allosteric Modulator of Beta2-adrenoceptor from a Natural Product-Derived DNA-Encoded Chemical Library.

Allosteric ligands are promising drugs owing to their remote regulations of the orthosteric ligand signaling pathway. There are few allosteric ligands due to the lack of handy and efficacious method for the screening. Herein, we developed an affinity chromatographic method for allosteric ligand screening by immobilizing purified beta2 adrenoceptor (β2-AR) onto macroporous silica gel by a two-point tethering method. The method relies on the occupation of the orthosteric site by an antagonist and the chelation of N-terminal His-tag of the receptor and Ni2+ coated on the gel. The immobilized β2-AR demonstrated the greatest allosteric responsive feature when Cmpd-15 (0.25 μM) was included in the mobile phase. Under the same conditions, the association constants of three agonists (salbutamol, terbutaline, and tulobuterol) reduced to 47%, 19%, and 27% compared with the data without the inclusion of Cmpd-15 in the mobile phase. APF was screened as a potential allosteric modulator of β2-AR by applying the immobilized receptor in a natural product-derived DNA-encoded chemical library (DEL). Relying on these results, we reasoned that the current method has potential in screening allosteric ligands of the receptor. We expect that it is applicable for the discovery of new allosteric binding sites of a target protein and screening allosteric modulators of the other receptors from complex samples.

Portability of Small Molecule Binding Sites in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure. Because they are associated with a range of diseases, IDPs are desirable therapeutic targets and have been targeted by small molecules. The transcription factor c‐Myc (Myc) is an IDP that is implicated in over 70% of cancers. Previously, we have shown that several small molecules can bind to short, contiguous sequences on Myc. These sequences remain unstructured when binding to small molecules, potentially indicating only a limited dependence on residues outside of the contiguous binding residues. This mode of binding differs from that common in structured proteins where contact residues are often noncontiguous and scaffolded by extensive protein structure. Here we test the portability of a small molecule‐IDP interaction implied by its dependence on only a short, linear sequence. We compare the binding of a small molecule to its specific recognition sequence in a native Myc context, in a minimalist peptide context, and by porting the binding sequence into the context of a different IDP. An ability of short, disordered sequences to bind small molecules in a context agnostic manner would have implications for the prediction and discovery of IDP binding sites broadly.

Thyroid hormone and thyroid hormone nuclear receptors: History and present state of art.

The present review traces the road leading to discovery of L-thyroxine, thyroid hormone (3,5,3´-triiodo-L-thyronine, T3) and its cognate nuclear receptors. Thyroid hormone is a pleio-tropic regulator of growth, differentiation, and tissue homeostasis in higher organisms. The major site of the thyroid hormone action is predominantly a cell nucleus. T3 specific binding sites in the cell nuclei have opened a new era in the field of the thyroid hormone receptors (TRs) discovery. T3 actions are mediated by high affinity nuclear TRs, TRalpha and TRbeta, which function as T3-activated transcription factors playing an essential role as transcription-modulating proteins affecting the transcriptional responses in target genes. Discovery and characterization of nuclear retinoid X receptors (RXRs), which form with TRs a heterodimer RXR/TR, positioned RXRs at the epicenter of molecular endocrinology. Transcriptional control via nuclear RXR/TR heterodimer represents a direct action of thyroid hormone. T3 plays a crucial role in the development of brain, it exerts significant effects on the cardiovascular system, skeletal muscle contractile function, bone development and growth, both female and male reproductive systems, and skin. It plays an important role in maintaining the hepatic, kidney and intestine homeostasis and in pancreas, it stimulates the beta-cell proliferation and survival. The TRs cross-talk with other signaling pathways intensifies the T3 action at cellular level. The role of thyroid hormone in human cancers, acting via its cognate nuclear receptors, has not been fully elucidated yet. This review is aimed to describe the history of T3 receptors, starting from discovery of T3 binding sites in the cell nuclei to revelation of T3 receptors as T3-inducible transcription factors in relation to T3 action at cellular level. It also focuses on milestones of investigation, comprising RXR/TR dimerization, cross-talk between T3 receptors, and other regulatory pathways within the cell and mainly on genomic action of T3. This review also focuses on novel directions of investigation on relationships between T3 receptors and cancer. Based on the update of available literature and the author's experimental experience, it is devoted to clinicians and medical students.

Characterization of a Bacillus megaterium strain with metal bioremediation potential and in silico discovery of novel cadmium binding motifs in the regulator, CadC.

Bioremediation of toxic metal ions using bacterial strains is a promising tool. Metal binding motifs in microbial proteins are involved in the regulation and transport of such toxic metals for metal detoxification. A bacterial strain designated TWSL_4 with metal (Cu, Cd, and Pb) resistance and removal ability was isolated and identified as a Bacillus megaterium strain using 16S rRNA gene analysis. An operon with 2 open reading frames (ORFs) was identified, cloned, and sequenced. ORF1 and ORF2 were identical to the cadmium efflux system accessory protein (CadC) and cadmium-translocating P-type ATPases (CadA) of B. megaterium strain YC4-R4 respectively. A protein homology search using Swiss model retrieved no crystal structures for CadC and CadA of Bacillus sp.. CadC of TWSL_4 had a sequence identity of 53% to the CadC (121aa) protein and 51.69% to the CadC crystal structure (1U2W.1.B; GMQE=0.75) of Staphylococcus sp. pI258. Molecular dynamic simulation studies revealed the presence of three metal binding regions in CadC of TWSL_4, [ASP7-TYR9], [ASP100-HIS102], and [LYS113-ASP116]. This is the first report showing evidence for the presence of Cd2+ and Zn2+ metal binding motifs in the CadC regulator of the Bacillus megaterium cad operon. The bacterial strain TWSL_4 was also found to contain two different P type ATPases encoding genes, cadA and zosA involved in metal resistance. Furthermore, the metal bioremediation potential of strain TWSL_4 was confirmed using an industrial effluent. KEY POINTS: • Isolation of a metal-resistant bacterial strain with potential for industrial bioremediation. • Discovery of novel Cd binding sites in CadC of the cad operon from B. megaterium. • Involvement of aspartic acid in the coordination of metal ions (Cd2+).

Recent advances in the design of choline kinase α inhibitors and the molecular basis of their inhibition.

Upregulated choline metabolism, characterized by an increase in phosphocholine (PCho), is a hallmark of oncogenesis and tumor progression. Choline kinase (ChoK), the enzyme responsible for PCho synthesis, has consequently become a promising drug target for cancer therapy and as such a significant number of ChoK inhibitors have been developed over the last few decades. More recently, due to the role of this enzyme in other pathologies, ChoK inhibitors have also been used in new therapeutic approaches against malaria and rheumatoid arthritis. Here, we review research results in the field of ChoKα inhibitors from their synthesis to the molecular basis of their binding mode. Strategies for the development of inhibitors and their selectivity on ChoKα over ChoKβ, the plasticity of the choline-binding site, the discovery of new exploitable binding sites, and the allosteric properties of this enzyme are highlighted. The outcomes summarized in this review will be a useful guide to develop new multifunctional potent drugs for the treatment of various human diseases.

A Binding Site Hotspot Map of the FKBP12-Rapamycin-FRB Ternary Complex by Photoaffinity Labeling and Mass Spectrometry-Based Proteomics.

Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photoaffinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12-photo-rapamycin-FRB ternary complex formed readily in vitro. Photoirradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 Å distance constraint between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photoactivation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the "undruggable" proteome.