Deciphering Phenotypes from Protein Biomarkers for Translational Research with PIPER.

Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has widespread clinical use for detection of inborn errors of metabolism, therapeutic drug monitoring, and numerous other applications. This technique detects proteolytic peptides as surrogates for protein biomarker expression, mutation, and post-translational modification in individual clinical assays and in cancer research with highly multiplexed quantitation across biological pathways. LC-MRM for protein biomarkers must be translated from multiplexed research-grade panels to clinical use. LC-MRM panels provide the capability to quantify clinical biomarkers and emerging protein markers to establish the context of tumor phenotypes that provide highly relevant supporting information. An application to visualize and communicate targeted proteomics data will empower translational researchers to move protein biomarker panels from discovery to clinical use. Therefore, we have developed a web-based tool for targeted proteomics that provides pathway-level evaluations of key biological drivers (e.g., EGFR signaling), signature scores (representing phenotypes) (e.g., EMT), and the ability to quantify specific drug targets across a sample cohort. This tool represents a framework for integrating summary information, decision algorithms, and risk scores to support Physician-Interpretable Phenotypic Evaluation in R (PIPER) that can be reused or repurposed by other labs to communicate and interpret their own biomarker panels.

Cereblon harnesses Myc-dependent bioenergetics and activity of CD8+ T lymphocytes.

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known "neosubstrates," such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon.

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.

Exploring the limits of the usefulness of mutagenesis in studies of allosteric mechanisms.

The outcome of structure-guided mutational analyses is often used in support of postulated mechanisms of protein allostery. However, the limits of how informative mutations can be in understanding allosteric mechanisms are not completely clear. Here, we report an exercise to evaluate whether mutational data can support a simplistic mechanistic model, developed with minimal data inputs. Due to the lack of a mechanism to explain how alanine allosterically modifies the affinity of human liver pyruvate kinase (approved symbol PKLR) for its substrate, phosphoenolpyruvate, we proposed a speculative allosteric mechanism for this system. Within the allosteric amino-acid-binding site (something in the effector site must, of necessity, contribute to the allosteric mechanism), we implemented multiple mutational strategies: (1) site-directed random mutagenesis at positions that contact bound alanine and (2) mutations to probe specific questions. Despite acknowledged inadequacies used to formulate the speculative mechanism, many mutations modified the allosteric coupling constant (Qax ) consistent with that mechanism. The observed support for this speculative mechanism leaves us to ponder the best use of mutational data in structure-function studies of allosteric mechanisms. The mutational databank derived from this exercise has an independent value for training and testing algorithms specific to allostery.

RWD Domain as an E2 (Ubc9)-Interaction Module.

An RWD domain is a well conserved domain found through bioinformatic analysis of the human proteome sequence; however, its function has been unknown. Ubiquitin-like modifications require the catalysis of three enzymes generally known as E1, E2, and E3. We solved the crystal structure of the E2 for the small ubiquitin-like modifiers (SUMO) in complex with an RWD domain and confirmed the structure using solution NMR analysis. The binding surface of RWD on Ubc9 is located near the N terminus of Ubc9 that is known to be involved in noncovalent binding of the proteins in the conjugation machinery, including a domain of E1, SUMO, and an E3 ligase. NMR data indicate that the RWD domain does not bind to SUMO and E1. The interaction between RWD and Ubc9 has a Kd of 32 ± 4 µM. Consistent with the structure and binding affinity and in contrast to a previous report, the RWD domain and RWDD3 have minimal effects on global SUMOylation. The structural and biochemical information presented here forms the basis for further investigation of the functions of RWD-containing proteins.

Implementation of a High-Accuracy Targeted Gene Expression Panel for Clinical Care.

This study describes the validation of a clinical RNA expression panel with evaluation of concordance between gene copy gain by a next-generation sequencing (NGS) assay and high gene expression by an RNA expression panel. The RNA Salah Targeted Expression Panel (RNA STEP) was designed with input from oncologists to include 204 genes with utility for clinical trial prescreening and therapy selection. RNA STEP was validated with the nanoString platform using remnant formalin-fixed, paraffin-embedded-derived RNA from 102 patients previously tested with a validated clinical NGS panel. The repeatability, reproducibility, and concordance of RNA STEP results with NGS results were evaluated. RNA STEP demonstrated high repeatability and reproducibility, with excellent correlation (r > 0.97, P < 0.0001) for all comparisons. Comparison of RNA STEP high gene expression (log2 ratio ≥ 2) versus NGS DNA-based gene copy number gain (copies ≥ 5) for 38 mutually covered genes revealed an accuracy of 93.0% with a positive percentage agreement of 69.4% and negative percentage agreement of 93.8%. Moderate correlation was observed between platforms (r = 0.53, P < 0.0001). Concordance between high gene expression and gene copy number gain varied by specific gene, and some genes had higher accuracy between assays. Clinical implementation of RNA STEP provides gene expression data complementary to NGS and offers a tool for prescreening patients for clinical trials.

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes.

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM(1)-PYK). To detail similarities/differences in the allosteric function of these two homologues, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM(1)-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM(1)-PYK (i.e., the l-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However, different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs rM(1)-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologues of a protein family.

Kinetic and spectroscopic studies of hemin acquisition in the hemophore HasAp from Pseudomonas aeruginosa.

The extreme limitation of free iron has driven various pathogens to acquire iron from the host in the form of heme. Specifically, several Gram-negative pathogens secrete a heme binding protein known as HasA to scavenge heme from the extracellular environment and to transfer it to the receptor protein HasR for import into the bacterial cell. Structures of heme-bound and apo-HasA homologues show that the heme iron(III) ligands, His32 and Tyr75, reside on loops extending from the core of the protein and that a significant conformational change must occur at the His32 loop upon heme binding. Here, we investigate the kinetics of heme acquisition by HasA from Pseudomonas aeruginosa (HasAp). The rate of heme acquisition from human met-hemoglobin (met-Hb) closely matches that of heme dissociation which suggests a passive mode of heme uptake from this source. The binding of free hemin is characterized by an initial rapid phase forming an intermediate before further conversion to the final complex. Analysis of this same reaction using an H32A variant lacking the His heme ligand shows only the rapid phase to form a heme-protein complex spectroscopically equivalent to that of the wild-type intermediate. Further characterization of these reactions using electron paramagnetic resonance and resonance Raman spectroscopy of rapid freeze quench samples provides support for a model in which heme is initially bound by the Tyr75 to form a high-spin heme-protein complex before slower coordination of the His32 ligand upon closing of the His loop over the heme. The slow rate of this loop closure implies that the induced-fit mechanism of heme uptake in HasAp is not based on a rapid sampling of the H32 loop between open and closed configurations but, rather, that the H32 loop motions are triggered by the formation of the high-spin heme-HasAp intermediate complex.

Structural, NMR spectroscopic, and computational investigation of hemin loading in the hemophore HasAp from Pseudomonas aeruginosa.

When challenged by low-iron conditions several Gram-negative pathogens secrete a hemophore (HasA) to scavenge hemin from its host and deliver it to a receptor (HasR) on their outer membrane for internalization. Here we report results from studies aimed at probing the structural and dynamic processes at play in the loading of the apo-hemophore secreted by P. aeruginosa (apo-HasAp) with hemin. The structure of apo-HasAp shows a large conformational change in the loop harboring axial ligand His32 relative to the structure of holo-HasAp, whereas the loop bearing the other axial ligand, Tyr75, remains intact. To investigate the role played by the axial ligand-bearing loops in the process of hemin capture we investigated the H32A mutant, which was found to exist as a monomer in its apo-form and as a mixture of monomers and dimers in its holo-form. We obtained an X-ray structure of dimeric H32A holo-HasAp, which revealed that the two subunits are linked by cofacial interactions of two hemin molecules and that the conformation of the Ala32 loop in the dimer is identical to that exhibited by the His32 loop in wild type apo-HasAp. Additional data suggest that the conformation of the Ala32 loop in the dimer is mainly a consequence of dimerization. Hence, to investigate the effect of hemin loading on the topology of the His32 loop we also obtained the crystal structure of monomeric H32A holo-HasAp coordinated by imidazole (H32A-imidazole) and investigated the monomeric H32A HasAp and H32A-imidazole species in solution by NMR spectroscopy. The structure of H32A-imidazole revealed that the Ala32 loop attains a "closed" conformation nearly identical to that observed in wild type holo-HasAp, and the NMR investigations indicated that this conformation is maintained in solution. The NMR studies also highlighted conformational heterogeneity at the H32 loop hinges and in other key sections of the structure. Targeted molecular dynamics simulations allowed us to propose a possible path for the closing of the His32 loop upon hemin binding and identified molecular motions that are likely important in transmitting the presence of hemin in the Tyr75 loop to the His32 loop to initiate its closing. Importantly, residues implicated as undergoing motions in the computations are also observed as being dynamic by NMR. Taken together, these observations provide direct experimental evidence indicating that hemin loads onto the Tyr75 loop of apo-HasAp, which triggers the closing of the His32 loop.

Structural characterization of the hemophore HasAp from Pseudomonas aeruginosa: NMR spectroscopy reveals protein-protein interactions between Holo-HasAp and hemoglobin.

Pseudomonas aeruginosa secretes a 205 residue long hemophore (full-length HasAp) that is subsequently cleaved at the C'-terminal domain to produce mainly a 184 residue long truncated HasAp that scavenges heme [Letoffé, S., Redeker, V., and Wandersman, C. (1998) Mol. Microbiol. 28, 1223-1234]. HasAp has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The X-ray crystal structure of truncated HasAp revealed a polypeptide alphabeta fold and a ferriheme coordinated axially by His32 and Tyr75, with the side chain of His83 poised to accept a hydrogen bond from the Tyr75 phenolic acid group. NMR investigations conducted with full-length HasAp showed that the carboxyl-terminal tail (21 residues) is disordered and conformationally flexible. NMR spectroscopic investigations aimed at studying a complex between apo-HasAp and human methemoglobin were stymied by the rapid heme capture by the hemophore. In an effort to circumvent this problem NMR spectroscopy was used to monitor the titration of 15N-labeled holo-HasAp with hemoglobin. These studies allowed identification of a specific area on the surface of truncated HasAp, encompassing the axial ligand His32 loop that serves as a transient site of interaction with hemoglobin. These findings are discussed in the context of a putative encounter complex between apo-HasAp and hemoglobin that leads to efficient hemoglobin-heme capture by the hemophore. Similar experiments conducted with full-length 15N-labeled HasAp and hemoglobin revealed a transient interaction site in full-length HasAp similar to that observed in the truncated hemophore. The spectral perturbations observed while investigating these interactions, however, are weaker than those observed for the interactions between hemoglobin and truncated HasAp, suggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.

Observation of an E2 (Ubc9)-homodimer by crystallography.

Post-translational modifications by the small ubiquitin-like modifiers (SUMO), in particular the formation of poly-SUMO-2 and -3 chains, regulates essential cellular functions and its aberration leads to life-threatening diseases (Geoffroy and Hay, 2009) [1]. It was shown previously that the non-covalent interaction between SUMO and the conjugating enzyme (E2) for SUMO, known as Ubc9, is required for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. However, the structure of SUMO-Ubc9 non-covalent complex, by itself, could not explain how the poly-SUMO-2/3 chain forms and consequently a Ubc9 homodimer, although never been observed, was proposed for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. Here, we solved the crystal structure of a heterotrimer containing a homodimer of Ubc9 and the RWD domain from RWDD3. The asymmetric Ubc9 homodimer is mediated by the N-terminal region of one Ubc9 molecule and a surface near the catalytic Cys of the second Ubc9 molecule (Fig. 1A). This N-terminal surface of Ubc9 that is involved in the homodimer formation also interacts with the RWD domain, the ubiquitin-fold domain of the SUMO activating enzyme (E1), SUMO, and the E3 ligase, RanBP2 (Knipscheer et al., 2007; Tong et al.. 1997; Tatham et al., 2005; Reverter and Lima, 2005; Capili and Lima, 2007; Wang et al., 2009, 2010; Wang and Chen, 2010; Alontaga et al., 2015) [2], [3], [4], [5], [6], [7], [8], [9], [10]. The existence of the Ubc9 homodimer in solution is supported by previously published solution NMR studies of rotational correlation time and chemical shift perturbation (Alontaga et al., 2015; Yuan et al., 1999) [10], [11]. Site-directed mutagenesis and biochemical analysis suggests that this dimeric arrangement of Ubc9 is likely important for poly-SUMO chain formation (Fig. 1B and C). The asymmetric Ubc9 homodimer described for the first time in this work could provide the critical missing link in the poly-SUMO chain formation mechanism. The data presented here are related to the research article entitled, "RWD domain as an E2 (Ubc9) interaction module" (Alontaga et al., 2015) [10]. The data of the crystal structure has been deposited to RCSB protein data bank with identifier: 4Y1L.

Design of high-throughput screening assays and identification of a SUMO1-specific small molecule chemotype targeting the SUMO-interacting motif-binding surface.

Protein-protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multidisciplinary approach to identify small-molecule disruptors of protein-protein interactions that are mediated by SUMO (small ubiquitin-like modifier) proteins. SUMO modifications have emerged as a target with importance in treating cancer, neurodegenerative disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein-protein interactions can sensitize cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS) to identify inhibitors for SUMO-dependent protein-protein interactions. Using these assays, we have identified a nonpeptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein-protein interaction, despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface.

Breast-cancer-secreted miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis.

Reprogrammed glucose metabolism as a result of increased glycolysis and glucose uptake is a hallmark of cancer. Here we show that cancer cells can suppress glucose uptake by non-tumour cells in the premetastatic niche, by secreting vesicles that carry high levels of the miR-122 microRNA. High miR-122 levels in the circulation have been associated with metastasis in breast cancer patients, and we show that cancer-cell-secreted miR-122 facilitates metastasis by increasing nutrient availability in the premetastatic niche. Mechanistically, cancer-cell-derived miR-122 suppresses glucose uptake by niche cells in vitro and in vivo by downregulating the glycolytic enzyme pyruvate kinase. In vivo inhibition of miR-122 restores glucose uptake in distant organs, including brain and lungs, and decreases the incidence of metastasis. These results demonstrate that, by modifying glucose utilization by recipient premetastatic niche cells, cancer-derived extracellular miR-122 is able to reprogram systemic energy metabolism to facilitate disease progression.

Impaired p32 regulation caused by the lymphoma-prone RECQ4 mutation drives mitochondrial dysfunction.

Mitochondrial DNA (mtDNA) encodes proteins that are important for ATP biogenesis. Therefore, changes in mtDNA copy number will have profound consequences on cell survival and proliferation. RECQ4 DNA helicase participates in both nuclear DNA and mtDNA synthesis. However, the mechanism that balances the distribution of RECQ4 in the nucleus and mitochondria is unknown. Here, we show that RECQ4 forms protein complexes with Protein Phosphatase 2A (PP2A), nucleophosmin (NPM), and mitochondrial p32 in different cellular compartments. Critically, the interaction with p32 negatively controls the transport of both RECQ4 and its chromatin-associated replication factor, MCM10, from the nucleus to mitochondria. Amino acids that are deleted in the most common cancer-associated RECQ4 mutation are required for the interaction with p32. Hence, this RECQ4 mutant, which is no longer regulated by p32 and is enriched in the mitochondria, interacts with the mitochondrial replication helicase PEO1 and induces abnormally high levels of mtDNA synthesis.

Biochemical analysis of protein SUMOylation.

SUMOylation, the covalent attachment of Small Ubiquitin-like MOdifier (SUMO) polypeptides to other proteins, is among the most important post-translational modifications that regulate the functional properties of a large number of proteins. SUMOylation is broadly involved in cellular processes such as gene transcription, hormone response, signal transduction, DNA repair, and nuclear transport. SUMO modification has also been implicated in the pathogenesis of human diseases, such as cancer, neurodegenerative disorders, and viral infection. Attachment of a SUMO protein to another protein is carried out in multiple steps catalyzed by three enzymes. This unit describes and discusses the in vitro biochemical methods used for investigating each step of the SUMOylation process. In addition, a high-throughput screening protocol is included for the identification of inhibitors of SUMOylation.

The impact of ions on allosteric functions in human liver pyruvate kinase.

Experimental designs used to monitor the magnitude of an allosteric response can greatly influence observed values. We report here the impact of buffer, monovalent cation, divalent cation, and anion on the magnitude of the allosteric regulation of the affinity of human liver pyruvate kinase (hL-PYK) for substrate, phosphoenolpyruvate (PEP). The magnitudes of the allosteric activation by fructose-1,6-bisphosphate (Fru-1,6-BP) and the allosteric inhibition by alanine are independent of most, but not all buffers tested. However, these magnitudes are dependent on whether Mg(2+) or Mn(2+) is included as the divalent cation. In the presence of Mn(2+), any change in K(app-PEP) caused by Fru-1,6-BP is minimal. hL-PYK activity does not appear to require monovalent cation. Monovalent cation binding in the active site impacts PEP affinity with minimum influence on the magnitude of allosteric coupling. However, Na(+) and Li(+) reduce the magnitude of the allosteric response to Fru-1,6-BP, likely due to mechanisms outside of the active site. Which anion is used to maintain a constant monovalent cation concentration also influences the magnitude of the allosteric response. The value of determining the impact of ions on allosteric function can be appreciated by considering that representative structures used in comparative studies have often been determined using protein crystals grown in diverse buffer and salt conditions.

13C NMR spectroscopy of core heme carbons as a simple tool to elucidate the coordination state of ferric high-spin heme proteins.

Evidence is presented demonstrating that the magnitudes of the 13C chemical shifts originating from heme meso carbons provide a straightforward diagnostic tool to elucidate the coordination state of high-spin heme proteins and enzymes. Pentacoordinate high-spin heme centers exhibit 13C meso shifts centered at approximately 250 ppm, whereas their hexacoordinate counterparts exhibit 13C shifts centered at approximately -80 ppm. The relatively small spectral window (400 to -100 ppm) covering the meso-13C shifts, the relatively narrow lines of these resonances, and the availability of biosynthetic methods to prepare 13C-labeled heme (Rivera, M.; Walker, F. A. Anal. Biochem. 1995, 230, 295-302) make this approach practical. The theoretical basis for the distinct chemical shifts observed for meso carbons from hexacoordinate high-spin hemes relative to their pentacoordinate counterparts are now well understood (Cheng, R.-J.; Chen, P. Y.; Lovell, T.; Liu, T.; Noodleman, L.; Case, D. A. J. Am. Chem. Soc. 2003, 125, 6774-6783), which indicates that the magnitude of the meso-carbon chemical shifts can be used as a simple and reliable diagnostic tool for determining the coordination state of the heme active sites, independent of the nature of the proximal ligand. Proof of the principle for the 13C NMR spectroscopic approach is demonstrated using hexa- and pentacoordinate myoglobin. Subsequently, 13C NMR spectroscopy has been used to unambiguously determine that a recently discovered heme protein from Shigella dysenteriae (ShuT) is pentacoordinate.

Heme oxidation in a chimeric protein of the alpha-selective Neisseriae meningitidis heme oxygenase with the distal helix of the delta-selective Pseudomonas aeruginosa.

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.