A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

Effect of Ligands and Transducers on the Neurotensin Receptor 1 Conformational Ensemble.

Using a discrete, intracellular 19F nuclear magnetic resonance (NMR) probe on transmembrane helix 6 of the neurotensin receptor 1 (NTS1), we aim to understand how ligands and transducers modulate the receptor's structural ensemble in a solution. For apo NTS1, 19F NMR spectra reveal an ensemble of at least three conformational substates (one inactive and two active-like) in equilibrium that exchange on the millisecond to second timescale. Dynamic NMR experiments reveal that these substates follow a linear three-site exchange process that is both thermodynamically and kinetically remodeled by orthosteric ligands. As previously observed in other G protein-coupled receptors (GPCRs), the full agonist is insufficient to completely stabilize the active-like state. The inactive substate is abolished upon coupling to ß-arrestin-1 (ßArr1) or the C-terminal helix of Gαq, which comprises ≳60% of the GPCR/G protein interface surface area. Whereas ßArr1 exclusively selects for pre-existing active-like substates, the Gαq peptide induces a new substate. Both transducer molecules promote substantial line broadening of active-like states, suggesting contributions from additional microsecond to millisecond exchange processes. Together, our study suggests that (i) the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and (ii) the available static structures do not represent the entire conformational ensemble observed in a solution.

Expanding the genotype and phenotype spectrum of SYT1-associated neurodevelopmental disorder.

PURPOSE: Synaptotagmin-1 (SYT1) is a critical mediator of neurotransmitter release in the central nervous system. Previously reported missense SYT1 variants in the C2B domain are associated with severe intellectual disability, movement disorders, behavioral disturbances, and electroencephalogram abnormalities. In this study, we expand the genotypes and phenotypes and identify discriminating features of this disorder. METHODS: We describe 22 individuals with 15 de novo missense SYT1 variants. The evidence for pathogenicity is discussed, including the American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria, known structure-function relationships, and molecular dynamics simulations. Quantitative behavioral data for 14 cases were compared with other monogenic neurodevelopmental disorders. RESULTS: Four variants were located in the C2A domain with the remainder in the C2B domain. We classified 6 variants as pathogenic, 4 as likely pathogenic, and 5 as variants of uncertain significance. Prevalent clinical phenotypes included delayed developmental milestones, abnormal eye physiology, movement disorders, and sleep disturbances. Discriminating behavioral characteristics were severity of motor and communication impairment, presence of motor stereotypies, and mood instability. CONCLUSION: Neurodevelopmental disorder-associated SYT1 variants extend beyond previously reported regions, and the phenotypic spectrum encompasses a broader range of severities than initially reported. This study guides the diagnosis and molecular understanding of this rare neurodevelopmental disorder and highlights a key role for SYT1 function in emotional regulation, motor control, and emergent cognitive function.

TRACT revisited: an algebraic solution for determining overall rotational correlation times from cross-correlated relaxation rates.

Accurate rotational correlation times ([Formula: see text]) are critical for quantitative analysis of fast timescale NMR dynamics. As molecular weights increase, the classic derivation of [Formula: see text] using transverse and longitudinal relaxation rates becomes increasingly unsuitable due to the non-trivial contribution of remote dipole-dipole interactions to longitudinal relaxation. Derivations using cross-correlated relaxation experiments, such as TRACT, overcome these limitations but are erroneously calculated in 65% of the citing literature. Herein, we developed an algebraic solutions to the Goldman relationship that facilitate rapid, point-by-point calculations for straightforward identification of appropriate spectral regions where global tumbling is likely to be dominant. The rigid-body approximation of the Goldman relationship has been previously shown to underestimate TRACT-based rotational correlation time estimates. This motivated us to develop a second algebraic solution that employs a simplified model-free spectral density function including an order parameter term that could, in principle, be set to an average backbone S2 ≈ 0.9 to further improve the accuracy of [Formula: see text] estimation. These solutions enabled us to explore the boundaries of the Goldman relationship as a function of the H-N internuclear distance ([Formula: see text]), difference of the two principal components of the axially-symmetric 15N CSA tensor ([Formula: see text]), and angle of the CSA tensor relative to the N-H bond vector ([Formula: see text]). We hope our algebraic solutions and analytical strategies will increase the accuracy and application of the TRACT experiment.

15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins.

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131-294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine-proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

Chemokine cooperativity is caused by competitive glycosaminoglycan binding.

Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, in which multiple chemokines are secreted simultaneously.

Structure-based ligand discovery for the protein-protein interface of chemokine receptor CXCR4.

G-protein-coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein-protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC(50) values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein-protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.

Heparin oligosaccharides inhibit chemokine (CXC motif) ligand 12 (CXCL12) cardioprotection by binding orthogonal to the dimerization interface, promoting oligomerization, and competing with the chemokine (CXC motif) receptor 4 (CXCR4) N terminus.

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.

Discovery and characterization of a disulfide-locked C(2)-symmetric defensin peptide.

We report the discovery of HD5-CD, an unprecedented C2-symmetric ß-barrel-like covalent dimer of the cysteine-rich host-defense peptide human defensin 5 (HD5). Dimerization results from intermonomer disulfide exchange between the canonical α-defensin Cys(II)-Cys(IV) (Cys(5)-Cys(20)) bonds located at the hydrophobic interface. This disulfide-locked dimeric assembly provides a new element of structural diversity for cysteine-rich peptides as well as increased protease resistance, broad-spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen Acinetobacter baumannii.

Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways.

Chemokines and chemokine receptors are extensively and broadly involved in cancer metastasis. Previously, we demonstrated that epigenetic silencing of the chemokine CXCL12 sensitizes breast and colon cancer cells to endocrine signaling and metastasis to distant tissues. Yet, the precise mechanism whereby CXCL12 production by tumor cells regulates dissemination remains unclear. Here, we show that administration of CXCL12 extended survival of tumor-bearing mice by potently limiting metastasis of colorectal carcinoma or murine melanoma. Because secreted CXCL12 is a mixture of monomeric and dimeric species in equilibrium, oligomeric variants that either promote (monomer) or halt (dimer) chemotaxis were used to dissect the mechanisms interrupting carcinoma metastasis. Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited ß-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12 activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2, but only weakly, if at all, recruited arrestin, stimulated actin polymerization, or promoted chemotaxis. NMR analyses illustrated that CXCL12 monomers made specific contacts with CXCR4 that were lost following dimerization. Our results establish the potential for inhibiting CXCR4-mediated metastasis by administration of CXCL12. Chemokine-mediated migration and ß-arrestin responses did not dictate the antitumor effect of CXCL12. We conclude that cellular migration is tightly regulated by selective CXCR4 signaling evoked by unique interactions with distinct ligand quaternary structures.

Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide.

G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and Ctail (H8-Ctail) to investigate its structural stability, conformational dynamics, and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift-derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement NMR. Taken together, our studies reveal the H8-Ctail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.

Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: A combined SAXS and NMR study.

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8's oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein's functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.

Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1.

Targeting of the multifunctional enzyme apurinic/apyrimidinic endonuclease I/redox factor 1 (APE1) has produced small molecule inhibitors of both its endonuclease and redox activities. While one of the small molecules, the redox inhibitor APX3330, completed a Phase I clinical trial for solid tumors and a Phase II clinical trial for Diabetic Retinopathy/Diabetic Macular Edema, the mechanism of action for this drug has yet to be fully understood. Here, we demonstrate through HSQC NMR studies that APX3330 induces chemical shift perturbations (CSPs) of both surface and internal residues in a concentration-dependent manner, with a cluster of surface residues defining a small pocket on the opposite face from the endonuclease active site of APE1. Furthermore, APX3330 induces partial unfolding of APE1 as evidenced by a time-dependent loss of chemical shifts for approximately 35% of the residues within APE1 in the HSQC NMR spectrum. Notably, regions that are partially unfolded include adjacent strands within one of two beta sheets that comprise the core of APE1. One of the strands comprises residues near the N-terminal region and a second strand is contributed by the C-terminal region of APE1, which serves as a mitochondrial targeting sequence. These terminal regions converge within the pocket defined by the CSPs. In the presence of a duplex DNA substrate mimic, removal of excess APX3330 resulted in refolding of APE1. Our results are consistent with a reversible mechanism of partial unfolding of APE1 induced by the small molecule inhibitor, APX3330, defining a novel mechanism of inhibition.

Stabilization of pre-existing neurotensin receptor conformational states by ß-arrestin-1 and the biased allosteric modulator ML314.

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor's intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs - without dramatically altering the structural ensemble. ß-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A ß-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS1).

Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS1). We establish that the NTS1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination.

Solution structure of CCL21 and identification of a putative CCR7 binding site.

CCL21 is a human chemokine that recruits normal immune cells and metastasizing tumor cells to lymph nodes through activation of the G protein-coupled receptor CCR7. The CCL21 structure solved by NMR contains a conserved chemokine domain followed by an extended, unstructured C-terminus that is not typical of most other chemokines. A sedimentation equilibrium study showed CCL21 to be monomeric. Chemical shift mapping indicates that the CCR7 N-terminus binds to the N-loop and third ß-strand of CCL21's chemokine domain. Details of CCL21-receptor recognition may enable structure-based drug discovery of novel antimetastatic agents.

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions.

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.

NMR in the Analysis of Functional Chemokine Interactions and Drug Discovery.

The involvement of chemokines and chemokine receptors in a great variety of pathological indications underscores their utility as therapeutic targets. In general, chemokine-mediated migration and signaling requires three distinct interactions: self-association, glycosaminoglycan (GAG) binding, and activation of G protein-coupled receptors (GPCRs). Solution-state nuclear magnetic resonance (NMR) spectroscopy has long been used to determine the apo structure of chemokines and monitor complex formation; however, it has never contributed directly to drug discovery efforts that are traditionally focused on the previously inaccessible chemokine receptors. Our lab recently demonstrated that NMR structures can be successfully utilized to direct drug discovery against chemokines. The ease of collecting chemokine structural data coupled with the increased efficiency of structure-based drug discovery campaigns makes chemokine-directed therapies particularly attractive. In addition, recent advances in sample preparation, spectrometer hardware, and pulse program development are allowing researchers to examine interactions with previously inaccessible partners - including full-length chemokine receptors. These developments will facilitate exploration of novel ways to modulate chemokine activity using structure-guided drug discovery.

A method for selective 19 F-labeling absent of probe sequestration (SLAPS).

Fluorine (19 F) offers several distinct advantages for biomolecular nuclear magnetic resonance spectroscopy such as no background signal, 100% natural abundance, high sensitivity, and a large chemical shift range. Exogenous cysteine-reactive 19 F-probes have proven especially indispensable for characterizing large, challenging systems that are less amenable to other isotopic labeling strategies such as G protein-coupled receptors. As fluorine linewidths are inherently broad, limiting reactions with offsite cysteines is critical for spectral simplification and accurate deconvolution of component peaks-especially when analyzing systems with intermediate to slow timescale conformational exchange. Here, we uncovered noncovalent probe sequestration by detergent proteomicelles as a second source of offsite labeling when using the popular 19 F-probe BTFMA (2-bromo-N-(4-[trifluoromethyl]phenyl)acetamide). The chemical shift and relaxation rates of these unreacted 19 F-BTFMA molecules are insufficient to distinguish them from protein-conjugates, but they can be easily identified using mass spectrometry. We present a simple four-step protocol for Selective Labeling Absent of Probe Sequestration (SLAPS): physically disrupt cell membranes in the absence of detergent, incubate membranes with cysteine-reactive 19 F-BTFMA, remove excess unreacted 19 F-BTFMA molecules via ultracentrifugation, and finally solubilize in the detergent of choice. Our approach builds upon the in-membrane chemical modification method with the addition of one crucial step: removal of unreacted 19 F-probes by ultracentrifugation prior to detergent solubilization. SLAPS is broadly applicable to other lipophilic cysteine-reactive probes and membrane protein classes solubilized in detergent micelles or lipid mimetics.

Sulfotyrosine recognition as marker for druggable sites in the extracellular space.

Chemokine signaling is a well-known agent of autoimmune disease, HIV infection, and cancer. Drug discovery efforts for these signaling molecules have focused on developing inhibitors targeting their associated G protein-coupled receptors. Recently, we used a structure-based approach directed at the sulfotyrosine-binding pocket of the chemokine CXCL12, and thereby demonstrated that small molecule inhibitors acting upon the chemokine ligand form an alternative therapeutic avenue. Although the 50 members of the chemokine family share varying degrees of sequence homology (some as little as 20%), all members retain the canonical chemokine fold. Here we show that an equivalent sulfotyrosine-binding pocket appears to be conserved across the chemokine superfamily. We monitored sulfotyrosine binding to four representative chemokines by NMR. The results suggest that most chemokines harbor a sulfotyrosine recognition site analogous to the cleft on CXCL12 that binds sulfotyrosine 21 of the receptor CXCR4. Rational drug discovery efforts targeting these sites may be useful in the development of specific as well as broad-spectrum chemokine inhibitors.