Michael E. Hodsdon

Structural and Functional Basis of CXCL12 (Stromal Cell-derived Factor-1{alpha}) Binding to Heparin

CXCL12 (SDF-1α) and CXCR4 are critical for embryonic development and cellular migration in adults. These proteins are involved in HIV-1 infection, cancer metastasis, and WHIM disease. Sequestration and presentation of CXCL12 to CXCR4 by glycosaminoglycans (GAGs) is proposed to be important for receptor activation. Mutagenesis has identified CXCL12 residues that bind to heparin. However, the molecular details of this interaction have not yet been determined. Here we demonstrate that soluble heparin and heparan sulfate negatively affect CXCL12-mediated in vitro chemotaxis. We also show that a cluster of basic residues in the dimer interface is required for chemotaxis and is a target for inhibition by heparin. We present structural evidence for binding of an unsaturated heparin disaccharide to CXCL12 attained through solution NMR spectroscopy and x-ray crystallography. Increasing concentrations of the disaccharide altered the two-dimensional 1H-15N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to β-strands in the dimer interface. The second includes the amino-terminal loop and the α-helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His25, Lys27, and Arg41 of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala20, Arg21, Asn30, and Lys64. This is the first x-ray structure of a CXC class chemokine in complex with glycosaminoglycans. Based on the observation of two heparin binding sites, we propose a mechanism in which GAGs bind around CXCL12 dimers as they sequester and present CXCL12 to CXCR4.

Allosteric inhibition of macrophage migration inhibitory factor revealed by ibudilast

AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5‐phosphatase domain followed by an ASH and a RhoGAP‐like domain. Their divergent NH2‐terminal portions remain uncharacterized. We show that the NH2‐terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin‐coated pits, was earlier shown to contain another binding site for clathrin in its COOH‐terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2‐terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin‐binding site in OCRL maps to an unusual clathrin‐box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin‐dependent membrane trafficking.

Structure of the EF-hand domain of polycystin-2 suggests a mechanism for Ca2+-dependent regulation of polycystin-2 channel activity

The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca2+-permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720–797), a flexible linker (798–827), and an oligomeric coiled coil domain (828–895). We found that PC2-EF binds Ca2+ at a single site and undergoes Ca2+-dependent conformational changes, suggesting it is an essential element of Ca2+-sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca2+-bound PC2-EF. Human PC2-EF contains a divergent non-Ca2+-binding helix-loop-helix (HLH) motif packed against a canonical Ca2+-binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R1 and R2 relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca2+-bound states, consistent with a role for PC2-EF as a Ca2+-sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca2+-dependent protein interactions. We propose that Ca2+-sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca2+ dependence of PC2 channel activity by PC2-EF.

An intrinsically disordered C terminus allows the La protein to assist the biogenesis of diverse noncoding RNA precursors

The La protein binds the 3′ ends of many newly synthesized noncoding RNAs, protecting these RNAs from nucleases and influencing folding, maturation, and ribonucleoprotein assembly. Although 3′ end binding by La involves the N-terminal La domain and adjacent RNA recognition motif (RRM), the mechanisms by which La stabilizes diverse RNAs from nucleases and assists subsequent events in their biogenesis are unknown. Here we report that a conserved feature of La proteins, an intrinsically disordered C terminus, is required for the accumulation of certain noncoding RNA precursors and for the role of the Saccharomyces cerevisiae La protein Lhp1p in assisting formation of correctly folded pre-tRNA anticodon stems in vivo. Footprinting experiments using purified Lhp1p reveal that the C terminus is required to protect a pre-tRNA anticodon stem from chemical modification. Although the C terminus of Lhp1p is hypersensitive to proteases in vitro, it becomes protease-resistant upon binding pre-tRNAs, U6 RNA, or pre-5S rRNA. Thus, while high affinity binding to 3′ ends requires the La domain and RRM, a conformationally flexible C terminus allows La to interact productively with a diversity of noncoding RNA precursors. We propose that intrinsically disordered domains adjacent to well characterized RNA-binding motifs in other promiscuous RNA-binding proteins may similarly contribute to the ability of these proteins to influence the cellular fates of multiple distinct RNA targets.

Calcium-induced Conformational Changes in C-terminal Tail of Polycystin-2 Are Necessary for Channel Gating

Background: Polycystin-2, a calcium-permeable TRP channel, is mutated in autosomal dominant polycystic kidney disease. Results: Calcium binding by the polycystin-2 EF-hand domain induces discrete conformational and oligomerization state transitions that impact channel gating. Conclusion: Polycystin-2 channel activity is regulated by cytoplasmic calcium-induced conformational changes. Significance: These studies provide a structural and mechanistic understanding for the impact of calcium binding on channel regulation. Polycystin-2 (PC2) is a Ca2+-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca2+. PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca2+-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca2+ remains unknown. We propose that the PC2 EF-hand is a Ca2+ sensor required for channel gating. Consistent with this, Ca2+ binding causes a dramatic decrease in the radius of gyration (Rg) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca2+ concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (Dmax) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca2+ via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca2+ release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca2+ levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca2+-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.

Ayurvedic herbal medicine and lead poisoning

Although the majority of published cases of lead poisoning come from occupational exposures, some traditional remedies may also contain toxic amounts of lead. Ayurveda is a system of traditional medicine that is native to India and is used in many parts of world as an alternative to standard treatment regimens. Here, we report the case of a 58-year-old woman who presented with abdominal pain, anemia, liver function abnormalities, and an elevated blood lead level. The patient was found to have been taking the Ayurvedic medicine Jambrulin prior to presentation. Chemical analysis of the medication showed high levels of lead. Following treatment with an oral chelating agent, the patients symptoms resolved and laboratory abnormalities normalized. This case highlights the need for increased awareness that some Ayurvedic medicines may contain potentially harmful levels of heavy metals and people who use them are at risk of developing associated toxicities.

Pediatric Fatality Following Ingestion of Dinitrophenol: Postmortem Identification of a “Dietary Supplement”

Dinitrophenol, a chemical currently used as an insecticide, is known to uncouple mitochondrial oxidative phosphorylation. A component of explosives, it has also been used in the past as a food coloring and clothing dye. In the 1930s, physicians prescribed it for weight loss, but this practice was discontinued when reports of cataracts, deaths, and other adverse outcomes came to light. We describe in our report the overdose and fatality of a teenager who purchased the product as a weight loss dietary supplement by mail order. We also describe a laboratory method that allowed postmortem determination of the dinitrophenol concentration in the victims serum. Her death, despite prompt medical treatment, underscores the danger of dinitrophenol. The easy accessibility and apparent resurgent interest in dinitrophenol as a weight loss agent is extremely timely and troubling.

Two Independent Histidines, One in Human Prolactin and One in Its Receptor, Are Critical for pH-dependent Receptor Recognition and Activation.

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

Is There Structural Specificity in the Reversible Protein Aggregates That Are Stored in Secretory Granules

There are several steps that must occur for secretory granules to form: (1) Secretory proteins that make up the dense cores of the granules must be concentrated; (2) membrane proteins necessary for granule function must accumulate in the correct location; and (3) inappropriate membrane proteins and excess membrane must be removed. Reversible aggregation of secretory granule proteins provides a mechanism for concentrating and sorting these proteins. There is specificity in the way secretory granule proteins are treated in cells that make granules. The specificity has been shown in some cases to occur after the aggregation process, so that granules containing different aggregates function differently. An explanation could be that a property of the aggregate, such as a surface motif, might influence the accumulation of membrane proteins necessary for granule function. Such a conclusion implies that the aggregates are not amorphous but have structure. Use of NMR spectroscopy to investigate changes in the environment of amino acid residues in secretory granule proteins as they form oligomers by using 15N relaxation times might provide a means to determine which residues are specifically involved in aggregation.