Exploring 2-Sulfonylpyrimidine Warheads as Acrylamide Surrogates for Targeted Covalent Inhibition: A BTK Story.

Targeted covalent inhibitors (TCIs) directing cysteine have historically relied on a narrow set of electrophilic "warheads". While Michael acceptors remain at the forefront of TCI design strategies, they show variable stability and selectivity under physiological conditions. Here, we show that the 2-sulfonylpyrimidine motif is an effective replacement for the acrylamide warhead of Ibrutinib, for the inhibition of Bruton's tyrosine kinase. In a few iterations, we discovered new derivatives, which inhibit BTK both in vitro and in cellulo at low nanomolar concentrations, on par with Ibrutinib. Several derivatives also displayed good plasma stability and reduced off-target binding in vitro across 135 tyrosine kinases. This proof-of-concept study on a well-studied kinase/TCI system highlights the 2-sulfonylpyrimidine group as a useful acrylamide replacement. In the future, it will be interesting to investigate its wider potential for developing TCIs with improved pharmacologies and selectivity profiles across structurally related protein families.

Allosteric regulation of kinase activity.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states.

Full-length Bruton's tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology [PHTH] domain and proline-rich regions [PRR] contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveal only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. Cryo-electron microscopy (cryoEM) data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with phosphatidylinositol (3,4,5)-trisphosphate. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

Screening and Characterization of Allosteric Small Molecules Targeting Bruton's Tyrosine Kinase.

Bruton's Tyrosine Kinase (BTK) is a nonreceptor tyrosine kinase that belongs to the TEC family. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA) leading to an arrest in B-cell development. BTK is also a drug target for B-cell lymphomas that rely on an intact B-cell receptor signaling cascade for survival. All FDA approved drugs for BTK target the ATP binding site of the catalytic kinase domain, leading to potential adverse events due to off-target inhibition. In addition, acquired resistance mutations occur in a subset of patients, rendering available BTK inhibitors ineffective. Therefore, allosteric sites on BTK should be explored for drug development to target BTK more specifically and in combination with active site inhibitors. Virtual screening against nonactive site pockets and in vitro experiments resulted in a series of small molecules that bind to BTK outside of the active site. We characterized these compounds using biochemical and biophysical techniques and narrowed our focus to compound "C2". C2 activates full-length BTK and smaller multidomain BTK fragments but not the isolated kinase domain, consistent with an allosteric mode of action. Kinetic experiments reveal a C2-mediated decrease in Km and an increase in kcat leading to an overall increase in the catalytic efficiency of BTK. C2 is also capable of activating the BTK XLA mutants. These proof-of-principle data reveal that BTK can be targeted allosterically with small molecules, providing an alternative to active site BTK inhibitors.

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states.

Full-length BTK has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology (PHTH) domain and proline-rich regions (PRR) contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveals only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. CryoEM data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with PIP3. Membrane-induced dimerizationactivates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to transautophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

Structure of BTK kinase domain with the second-generation inhibitors acalabrutinib and tirabrutinib.

Bruton's tyrosine kinase (BTK) is the target of the therapeutic agent, Ibrutinib, that treats chronic lymphocyte leukemia (CLL), mantle cell lymphoma (MCL) and other B cell malignancies. Ibrutinib is a first in class, covalent BTK inhibitor that limits B-cell survival and proliferation. Designing new inhibitors of BTK has been an important objective for advancing development of improved therapeutic agents against cancer and autoimmune disorders. Based on the success of Ibrutinib, several second-generation irreversible BTK inhibitors have been developed that exhibit fewer off-target effects. However, the binding-mode and their interaction with Btk have not been experimentally determined and evaluated at atomic resolution. Here we determined the first crystal structure of the BTK kinase domain in complex with acalabrutinib. In addition, we report a structure of the BTK/tirabrutinib complex and compare these structures with previously solved structures. The structures provide insight in the superior selectivity reported for acalabrutinb and guide future BTK inhibitor development.

Impact of the clinically approved BTK inhibitors on the conformation of full-length BTK and analysis of the development of BTK resistance mutations in chronic lymphocytic leukemia.

Inhibition of Bruton's tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph, R.E., et al., 2020, https://doi.org/10.7554/eLife.60470 ). Here we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

Conformational switches that control the TEC kinase - PLCγ signaling axis.

Cell surface receptors such as the T-cell receptor (TCR) and B-cell receptor (BCR) engage with external stimuli to transmit information into the cell and initiate a cascade of signaling events that lead to gene expression that drives the immune response. At the heart of controlling T- and B-cell cell signaling, phospholipase Cγ hydrolyzes membrane associated PIP2, leading to generation of the second messengers IP3 and DAG. These small molecules trigger mobilization of intracellular Ca2+ and promote transcription factor transport into the nucleus launching the adaptive immune response. The TEC family kinases are responsible for phosphorylating and activating PLCγ, and our group aims to understand mechanisms that regulate immune cell signal transduction by focusing on this kinase/phospholipase axis in T-cells and B-cells. Here, we review the current molecular level understanding of how the TEC kinases (ITK and BTK) and PLCγ1/2 are autoinhibited prior to activation of cell surface receptors, how TEC kinases are activated to specifically recognize the PLCγ substrate, and how conformational changes induced by phosphorylation trigger PLCγ activation.

The Conformational State of the BTK Substrate PLCγ Contributes to Ibrutinib Resistance.

Mutations in PLCγ, a substrate of the tyrosine kinase BTK, are often found in patients who develop resistance to the BTK inhibitor Ibrutinib. However, the mechanisms by which these PLCγ mutations cause Ibrutinib resistance are unclear. Under normal signaling conditions, BTK mediated phosphorylation of Y783 within the PLCγ cSH2-linker promotes the intramolecular association of this site with the adjacent cSH2 domain resulting in active PLCγ. Thus, the cSH2-linker region in the center of the regulatory gamma specific array (γSA) of PLCγ is a key feature controlling PLCγ activity. Even in the unphosphorylated state this linker exists in a conformational equilibrium between free and bound to the cSH2 domain. The position of this equilibrium is optimized within the properly regulated PLCγ enzyme but may be altered in the context of mutations. We therefore assessed the conformational status of four resistance associated mutations within the PLCγ γSA and find that they each alter the conformational equilibrium of the γSA leading to a shift toward active PLCγ. Interestingly, two distinct modes of mutation induced activation are revealed by this panel of Ibrutinib resistance mutations. These findings, along with the recently determined structure of fully autoinhibited PLCγ, provide new insight into the nature of the conformational change that occurs within the γSA regulatory region to affect PLCγ activation. Improving our mechanistic understanding of how B cell signaling escapes Ibrutinib treatment via mutations in PLCγ will aid in the development of strategies to counter drug resistance.

Reining in BTK: Interdomain Interactions and Their Importance in the Regulatory Control of BTK.

Since Dr. Ogden Bruton's 1952 paper describing the first human primary immunodeficiency disease, the peripheral membrane binding signaling protein, aptly named Bruton's tyrosine kinase (BTK), has been the target of intense study. Dr. Bruton's description of agammaglobulinemia set the stage for ultimately understanding key signaling steps emanating from the B cell receptor. BTK is a multidomain tyrosine kinase and in the decades since Dr. Bruton's discovery it has become clear that genetic defects in the regulatory domains or the catalytic domain can lead to immunodeficiency. This finding underscores the intricate regulatory mechanisms within the BTK protein that maintain appropriate levels of signaling both in the resting B cell and during an immune challenge. In recent decades, BTK has become a target for clinical intervention in treating B cell malignancies. The survival reliance of B cell malignancies on B cell receptor signaling has allowed small molecules that target BTK to become essential tools in treating patients with hematological malignancies. The first-in-class Ibrutinib and more selective second-generation inhibitors all target the active site of the multidomain BTK protein. Therapeutic interventions targeting BTK have been successful but are plagued by resistance mutations that render drug treatment ineffective for some patients. This review will examine the molecular mechanisms that drive drug resistance, the long-range conformational effects of active site inhibitors on the BTK regulatory apparatus, and emerging opportunities to allosterically target the BTK kinase to improve therapeutic interventions using combination therapies.

Differential impact of BTK active site inhibitors on the conformational state of full-length BTK.

Bruton's tyrosine kinase (BTK) is targeted in the treatment of B-cell disorders including leukemias and lymphomas. Currently approved BTK inhibitors, including Ibrutinib, a first-in-class covalent inhibitor of BTK, bind directly to the kinase active site. While effective at blocking the catalytic activity of BTK, consequences of drug binding on the global conformation of full-length BTK are unknown. Here, we uncover a range of conformational effects in full-length BTK induced by a panel of active site inhibitors, including large-scale shifts in the conformational equilibria of the regulatory domains. Additionally, we find that a remote Ibrutinib resistance mutation, T316A in the BTK SH2 domain, drives spurious BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from the autoinhibited form. Future development of BTK inhibitors will need to consider long-range allosteric consequences of inhibitor binding, including the emerging application of these BTK inhibitors in treating COVID-19.

Treatments for blood cancers, such as leukemia and lymphoma, rely heavily on chemotherapy, using drugs that target a vulnerable aspect of the cancer cells. B-cells, a type of white blood cell that produces antibodies, require a protein called Bruton's tyrosine kinase, or BTK for short, to survive. The drug ibrutinib (Imbruvica) is used to treat B-cell cancers by blocking BTK. The BTK protein consists of several regions. One of them, known as the kinase domain, is responsible for its activity as an enzyme (which allows it to modify other proteins by adding a 'tag' known as a phosphate group). The other regions of BTK, known as regulatory modules, control this activity. In BTK's inactive form, the regulatory modules attach to the kinase domain, blocking the regulatory modules from interacting with other proteins. When BTK is activated, it changes its conformation so the regulatory regions detach and become available for interactions with other proteins, at the same time exposing the active kinase domain. Ibrutinib and other BTK drugs in development bind to the kinase domain to block its activity. However, it is not known how this binding affects the regulatory modules. Previous efforts to study how drugs bind to BTK have used a version of the protein that only had the kinase domain, instead of the full-length protein. Now, Joseph et al. have studied full-length BTK and how it binds to five different drugs. The results reveal that ibrutinib and another drug called dasatinib both indirectly disrupt the normal position of the regulatory domains pushing BTK toward a conformation that resembles the activated state. By contrast, the three other compounds studied do not affect the inactive structure. Joseph et al. also examined a mutation in BTK that confers resistance against ibrutinib. This mutation increases the activity of BTK by disrupting the inactive structure, leading to B cells surviving better. Understanding how drug resistance mechanisms can work will lead to better drug treatment strategies for cancer. BTK is also a target in other diseases such as allergies or asthma and even COVID-19. If interactions between partner proteins and the regulatory domain are important in these diseases, then they may be better treated with drugs that maintain the regulatory modules in their inactive state. This research will help to design drugs that are better able to control BTK activity.

Lipid-targeting pleckstrin homology domain turns its autoinhibitory face toward the TEC kinases.

The pleckstrin homology (PH) domain is well known for its phospholipid targeting function. The PH-TEC homology (PHTH) domain within the TEC family of tyrosine kinases is also a crucial component of the autoinhibitory apparatus. The autoinhibitory surface on the PHTH domain has been previously defined, and biochemical investigations have shown that PHTH-mediated inhibition is mutually exclusive with phosphatidylinositol binding. Here we use hydrogen/deuterium exchange mass spectrometry, nuclear magnetic resonance (NMR), and evolutionary sequence comparisons to map where and how the PHTH domain affects the Bruton's tyrosine kinase (BTK) domain. The data map a PHTH-binding site on the activation loop face of the kinase C lobe, suggesting that the PHTH domain masks the activation loop and the substrate-docking site. Moreover, localized NMR spectral changes are observed for non-surface-exposed residues in the active site and on the distal side of the kinase domain. These data suggest that the association of PHTH induces allosteric conformational shifts in regions of the kinase domain that are critical for catalysis. Through statistical comparisons of diverse tyrosine kinase sequences, we identify residues unique to BTK that coincide with the experimentally determined PHTH-binding surface on the kinase domain. Our data provide a more complete picture of the autoinhibitory conformation adopted by full-length TEC kinases, creating opportunities to target the regulatory domains to control the function of these kinases in a biological setting.

The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell-specific adapter protein.

T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.

Dynamic regulatory features of the protein tyrosine kinases.

The SRC, Abelson murine leukemia viral oncogene homolog 1, TEC and C-terminal SRC Kinase families of non-receptor tyrosine kinases (collectively the Src module kinases) mediate an array of cellular signaling processes and are therapeutic targets in many disease states. Crystal structures of Src modules kinases provide valuable insights into the regulatory mechanisms that control activation and generate a framework from which drug discovery can advance. The conformational ensembles visited by these multidomain kinases in solution are also key features of the regulatory machinery controlling catalytic activity. Measurement of dynamic motions within kinases substantially augments information derived from crystal structures. In this review, we focus on a body of work that has transformed our understanding of non-receptor tyrosine kinase regulation from a static view to one that incorporates how fluctuations in conformational ensembles and dynamic motions influence activation status. Regulatory dynamic networks are often shared across and between kinase families while specific dynamic behavior distinguishes unique regulatory mechanisms for select kinases. Moreover, intrinsically dynamic regions of kinases likely play important regulatory roles that have only been partially explored. Since there is clear precedence that kinase inhibitors can exploit specific dynamic features, continued efforts to define conformational ensembles and dynamic allostery will be key to combating drug resistance and devising alternate treatments for kinase-associated diseases.

Multidomain Control Over TEC Kinase Activation State Tunes the T Cell Response.

Signaling through the T cell antigen receptor (TCR) activates a series of tyrosine kinases. Directly associated with the TCR, the SRC family kinase LCK and the SYK family kinase ZAP-70 are essential for all downstream responses to TCR stimulation. In contrast, the TEC family kinase ITK is not an obligate component of the TCR cascade. Instead, ITK functions as a tuning dial, to translate variations in TCR signal strength into differential programs of gene expression. Recent insights into TEC kinase structure have provided a view into the molecular mechanisms that generate different states of kinase activation. In resting lymphocytes, TEC kinases are autoinhibited, and multiple interactions between the regulatory and kinase domains maintain low activity. Following TCR stimulation, newly generated signaling modules compete with the autoinhibited core and shift the conformational ensemble to the fully active kinase. This multidomain control over kinase activation state provides a structural mechanism to account for ITK's ability to tune the TCR signal.

Achieving a Graded Immune Response: BTK Adopts a Range of Active/Inactive Conformations Dictated by Multiple Interdomain Contacts.

Capturing the functionally relevant forms of dynamic, multidomain proteins is extremely challenging. Bruton's tyrosine kinase (BTK), a kinase essential for B and mast cell function, has stubbornly resisted crystallization in its full-length form. Here, nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry show that BTK adopts a closed conformation in dynamic equilibrium with open, active conformations. BTK lacks the phosphotyrosine regulatory tail of the SRC kinases, yet nevertheless achieves a phosphotyrosine-independent C-terminal latch. The unique proline-rich region is an internal "on" switch pushing the autoinhibited kinase toward its active state. Newly identified autoinhibitory contacts in the BTK pleckstrin homology domain are sensitive to phospholipid binding, which induces large-scale allosteric changes. The multiplicity of these regulatory contacts suggests a clear mechanism for gradual or "analog" kinase activation as opposed to a binary "on/off" switch. The findings illustrate how previously modeled information for recalcitrant full-length proteins can be expanded and validated with a convergent multidisciplinary experimental approach.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP3. By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.