Structure-function analysis of cysteine residues in the plasmodium falciparum chitinase, PfCHT1.

The Plasmodium ookinete uses chitinase activity to penetrate the acellular, chitin-containing peritrophic matrix to invade the mosquito vector. Plasmodium ookinetes from different parasite clades secrete two structurally distinct forms of chitinase, one, a short form lacking a C-terminal putative chitin-binding domain (CBD), the other, a long form with both proenzyme and C-terminal putative chitin-binding domains. Here, we structurally and functionally characterize the three cysteines in the short chitinase of the human-infecting malaria parasite, P. falciparum testing the hypothesis that one unpaired cysteine would not contribute to chitinase-specific enzymatic activity which would identify this residue as potentially involved in intermolecular disulfide bonding and heteromultimeric invasion complex formation as previously described. To test this hypothesis, we produced and characterized recombinant wild-type and cysteine-mutation PfCHT1 proteins in E. coli and used biophysical and enzymatic approaches to examine their enzymatic activities and chitin-binding affinities. The cysteine-203 PfCHT1 mutation had no effect on chitinolytic and chitin-binding functions. The cysteine-220 and cysteine-230 mutants were enzymatically inactive and did not bind to chitin. The artificial intelligence-based protein prediction algorithm, AlphaFold, correctly identified the involvement of cys-220 and cys-230 in the intramolecular disulfide linkages key to maintaining properly folded chitinase structural integrity. AlphaFold predicted that cys-203 cysteine is surface exposed and thus involved in intermolecular protein-protein interaction. Production of the cys-to-ser 203 PfCHT1 mutant facilitated recombinant protein production. Future cellular and biochemical studies are needed to further understand details of Plasmodium ookinete mosquito midgut invasion.

A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D-dopachrome tautomerase.

The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.

The N-terminal length and side-chain composition of CXCL13 affect crystallization, structure and functional activity.

CXCL13 is the cognate chemokine agonist of CXCR5, a class A G-protein-coupled receptor (GPCR) that is essential for proper humoral immune responses. Using a `methionine scanning' mutagenesis method on the N-terminus of CXCL13, which is the chemokine signaling region, it was shown that minor length alterations and side-chain substitutions still result in CXCR5 activation. This observation indicates that the orthosteric pocket of CXCR5 can tolerate these changes without severely affecting the activity. The introduction of bulk on the ligand was well tolerated by the receptor, whereas a loss of contacts was less tolerated. Furthermore, two crystal structures of CXCL13 mutants were solved, both of which represent the first uncomplexed structures of the human protein. These structures were stabilized by unique interactions formed by the N-termini of the ligands, indicating that CXCL13 exhibits substantial N-terminal flexibility while the chemokine core domain remains largely unchanged. Additionally, it was observed that CXCL13 harbors a large degree of flexibility in the C-terminal extension of the ligand. Comparisons with other published structures of human and murine CXCL13 validate the relative rigidity of the core domain as well as the N- and C-terminal mobilities. Collectively, these mutants and their structures provide the field with additional insights into how CXCL13 interacts with CXCR5.

High-Throughput Screening of a Functional Human CXCL12-CXCR4 Signaling Axis in a Genetically Modified S. cerevisiae: Discovery of a Novel Up-Regulator of CXCR4 Activity.

CXCL12 activates CXCR4 and is involved in embryogenesis, hematopoiesis, and angiogenesis. It has pathological roles in HIV-1, WHIM disease, cancer, and autoimmune diseases. An antagonist, AMD3100, is used for the release of CD34+ hematopoietic stem cells from the bone marrow for autologous transplantation for lymphoma or multiple myeloma patients. Adverse effects are tolerated due to its short-term treatment, but AMD3100 is cardiotoxic in clinical studies for HIV-1. In an effort to determine whether Saccharomyces cerevisiae expressing a functional human CXCR4 could be used as a platform for identifying a ligand from a library of less ∼1,000 compounds, a high-throughput screening was developed. We report that 2-carboxyphenyl phosphate (fosfosal) up-regulates CXCR4 activation only in the presence of CXCL12. This is the first identification of a compound that increases CXCR4 activity by any mechanism. We mapped the fosfosal binding site on CXCL12, described its mechanism of action, and studied its chemical components, salicylate and phosphate, to conclude that they synergize to achieve the functional effect.

Antioxidant enzyme cycling over reproductive lunar cycles in Pocillopora damicornis.

The impacts of continued degradation of watersheds on coastal coral reefs world-wide is alarming, and action addressing anthropogenic stressors and subsequent rehabilitation of watersheds and adjacent reefs is an urgent priority. The aim of this study is to develop and improve the use of antioxidant enzymes as bioindicators of stress in coral species. In order to fully develop such tools, it is necessary to first understand baseline cycling of these enzymes within coral tissues. Due to inherent links between reproduction and oxidative stress, these aims may be facilitated by sampling coral tissues over reproductively-linked lunar cycles to determine variations from baseline. By developing a greater understanding of biochemical markers of stress in corals, specifically antioxidant defense enzymes catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in Hawaiian Pocillopora damicornis, we have provided molecular tools that identify thresholds of stress on coral reefs. Our results suggest that the coral reproductive state is a significant factor affecting the activity of antioxidant enzymes. Specifically, CAT and GR display maximum activity during peak reproductive state. Whereas significant maximal Se-independent GPx and SOD activity was measured during off-peak reproductive cycles. Such insight into the cyclical variation of the activity of these enzymes should be applied towards differentiating the influence of natural biological activity cycling in diagnostic tests identifying the effects of different physical environmental factors and chemical pollutants on coral health. Through the development and application of these molecular biomarkers of stress, we look to improve our ability to identify problems at the sub-lethal level, when action can be taken to mitigate a/biotic impacts.

Characterization, Dynamics, and Mechanism of CXCR4 Antagonists on a Constitutively Active Mutant.

The G protein-coupled receptor (GPCR) CXCR4 is a co-receptor for HIV and is involved in cancers and autoimmune diseases. We characterized five purine or quinazoline core polyamine pharmacophores used for targeting CXCR4 dysregulation in diseases. All were neutral antagonists for wild-type CXCR4 and two were biased antagonists with effects on ß-arrestin-2 only at high concentrations. These compounds displayed various activities for a constitutively active mutant (CAM). We use the IT1t-CXCR4 crystal structure and molecular dynamics (MD) simulations to develop two hypotheses for the activation of the N1193.35A CAM. The N1193.35A mutation facilitates increased coupling of TM helices III and VI. IT1t deactivates the CAM by disrupting the coupling between TM helices III and VI, mediated primarily by residue F872.53. Mutants of F872.53 in N1193.35A CXCR4 precluded constitutive signaling and prevented inverse agonism. This work characterizes CXCR4 ligands and provides a mechanism for N1193.35A constitutive activation.

The repeat region of cortactin is intrinsically disordered in solution.

The multi-domain protein, cortactin, contains a 37-residue repeating motif that binds to actin filaments. This cortactin repeat region comprises 6½ similar copies of the motif and binds actin filaments. To better understand this region of cortactin, and its fold, we conducted extensive biophysical analysis. Size exclusion chromatography with multi-angle light scattering (SEC-MALS) reveals that neither constructs of the cortactin repeats alone or together with the adjacent helical region homo-oligomerize. Using circular dichroism (CD) we find that in solution the cortactin repeats resemble a coil-like intrinsically disordered protein. Small-angle X-ray scattering (SAXS) also indicates that the cortactin repeats are intrinsically unfolded, and the experimentally observed radius of gyration (R g) is coincidental to that calculated by the program Flexible-Meccano for an unfolded peptide of this length. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the domain contains limited hydrophobic core regions. These experiments therefore provide evidence that in solution the cortactin repeat region of cortactin is intrinsically disordered.

Changes to coral health and metabolic activity under oxygen deprivation.

On Hawaiian reefs, the fast-growing, invasive algae Gracilaria salicornia overgrows coral heads, restricting water flow and light, thereby smothering corals. Field data shows hypoxic conditions (dissolved oxygen (DO2) < 2 mg/L) occurring underneath algal mats at night, and concurrent bleaching and partial tissue loss of shaded corals. To analyze the impact of nighttime oxygen-deprivation on coral health, this study evaluated changes in coral metabolism through the exposure of corals to chronic hypoxic conditions and subsequent analyses of lactate, octopine, alanopine, and strombine dehydrogenase activities, critical enzymes employed through anaerobic respiration. Following treatments, lactate and octopine dehydrogenase activities were found to have no significant response in activities with treatment and time. However, corals subjected to chronic nighttime hypoxia were found to exhibit significant increases in alanopine dehydrogenase activity after three days of exposure and strombine dehydrogenase activity starting after one overnight exposure cycle. These findings provide new insights into coral metabolic shifts in extremely low-oxygen environments and point to ADH and SDH assays as tools for quantifying the impact of hypoxia on coral health.

Structural studies of cerebral cavernous malformations 2 (CCM2) reveal a folded helical domain at its C-terminus.

Cerebral cavernous malformations (CCM) are neurovascular dysplasias affecting up to 0.5% of the population. Mutations in the CCM2 gene are associated with acquisition of CCM. We identify a previously uncharacterized domain at the C-terminus of CCM2 and determine its 1.9Å resolution crystal structure. Because this domain is structurally homologous to the N-terminal domain of harmonin, we name it the CCM2 harmonin-homology domain or HHD. CCM2 HHD is observed in two conformations, and we employ analytical ultracentrifugation to test its oligomerization. Additionally, CCM2 HHD contains an unusually long 13-residue 3(10) helix. This study provides the first structural characterization of CCM2.

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.

Kinetic characterization of Salmonella FliK-FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch.

The bacterial flagellum is a complex macromolecular machine consisting of more than 20 000 proteins, most of which must be exported from the cell via a dedicated Type III secretion apparatus. At a defined point in flagellar morphogenesis, hook completion is sensed and the apparatus switches substrate specificity type from rod and hook proteins to filament ones. How the switch works is a subject of intense interest. FliK and FlhB play central roles. In the present study, two optical biosensing methods were used to characterize FliK-FlhB interactions using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. Binding was found to be complex with fast and slow association and dissociation components. Surprisingly, wild-type and variant FlhBs had similar kinetic profiles and apparent affinities, which ranged between 1 and 10.5 microM, suggesting that the specificity switch is more complex than presently understood. Other binding experiments provided evidence for a conformational change after binding. Liquid chromatography-mass spectrometry (LC-MS) and NMR experiments were performed to identify a cyclic intermediate product whose existence supports the mechanism of autocatalytic cleavage at FlhB residue N269. The present results show that while autocatalytic cleavage is necessary for proper substrate specificity switching, it does not result in an altered interaction with FliK, strongly suggesting the involvement of other proteins in the mechanism.

The structural basis of integrin-linked kinase-PINCH interactions.

The heterotrimeric complex between integrin-linked kinase (ILK), PINCH, and parvin is an essential signaling platform, serving as a convergence point for integrin and growth-factor signaling and regulating cell adhesion, spreading, and migration. We report a 1.6-A crystal structure of the ILK ankyrin repeat domain bound to the PINCH1 LIM1 domain, revealing the molecular basis of ILK-PINCH interactions and providing a structural description of this region of ILK. This structure identifies 5 ankyrin repeats in ILK, explains previous deletion mutagenesis data, permits identification of ILK and PINCH1 point mutations that disrupt the interaction, shows how zincs are coordinated by PINCH1 LIM1, and suggests that conformational flexibility and twisting between the 2 zinc fingers within the LIM1 domain may be important for ILK binding. These data provide an atomic-resolution description of a key interaction in the ILK-PINCH-parvin scaffolding complex.

Structural and functional basis of CXCL12 (stromal cell-derived factor-1 alpha) binding to heparin.

CXCL12 (SDF-1alpha) 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 (1)H-(15)N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to beta-strands in the dimer interface. The second includes the amino-terminal loop and the alpha-helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His(25), Lys(27), and Arg(41) of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala(20), Arg(21), Asn(30), and Lys(64). 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.

CD44 is the signaling component of the macrophage migration inhibitory factor-CD74 receptor complex.

The macrophage migration inhibitory factor (MIF) receptor (CD74) was cloned recently, but the signaling mechanism is not evident. We hypothesized that signaling requires an additional molecule such as CD44, which activates nonreceptor tyrosine kinases. We utilized the CD74- and CD44-deficient COS-7/M6 cell to create stable transfectants expressing CD74, CD44, and a truncated CD44 lacking its intracytoplasmic signaling domain. CD74 alone mediated MIF binding; however, MIF-induced ERK1 and ERK2 kinase phosphorylation required the coexpression of full-length CD44. MIF binding was associated with the serine phosphorylation of CD74 and CD44. Investigations that used siRNA or kinase inhibitors indicate that MIF-induced ERK1 and ERK2 activation through CD44 required the Src tyrosine kinase. Studies of CD74, CD44, and CD74-CD44 transformants and corresponding mutant cells showed that CD74 and CD44 were necessary for MIF protection from apoptosis. These data establish CD44 as an integral member of the CD74 receptor complex leading to MIF signal transduction.

The FliN-FliH interaction mediates localization of flagellar export ATPase FliI to the C ring complex.

FliH regulates the flagellar export ATPase FliI, preventing nonproductive ATP hydrolysis. FliH has been shown to stably associate with the C ring protein FliN. Analysis of this complex reveals that FliH is required for FliI localization to the C ring, and thus FliH not only inhibits FliI ATPase activity but also may act to target FliI to the basal body. Quantitative binding studies revealed a KD of 110 nM for FliH binding to FliN. The KD for FliH binding of a FliN variant from a temperature-sensitive nonflagellate fliN point mutant was determined to be 270 nM, suggesting a molecular explanation for its phenotype. Another variant FliN from a temperature-sensitive mutant with a different phenotype displayed binding with an intermediate affinity. Weak export activity in a fliN null mutant was greatly increased by overproduction of FliI, mimicking a previously observed FliH bypass effect and supporting the conclusion that FliN-FliH binding is important for localization of FliI to the C ring and thus the membrane-embedded export apparatus beyond. A model incorporating the present findings is presented.

Integrity of extracellular loop 1 of the human cannabinoid receptor 1 is critical for high-affinity binding of the ligand CP 55,940 but not SR 141716A.

Like other G-protein coupled receptors with hydrophobic ligands, the human cannabinoid receptor 1 (CB1) is thought to bind its ligands within the transmembrane region of the receptor. However, for some of these receptors the extracellular loops (ECs) have also been shown to play a role in ligand recognition and selectivity. We have taken a mutagenesis approach to examine the role of the amino terminus, EC1, and EC3 of CB1 in ligand binding. Eight mutant receptors, each with a dipeptide insertion, were constructed, expressed, and evaluated for binding to the cannabinoid ligands (-)-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). Mutants with insertions in the membrane distal region of the amino terminus or EC3 maintained affinity for both ligands. Those with insertions in the membrane proximal region of the amino terminus or EC1 exhibited a loss of affinity for CP 55,940 while retaining wild-type affinity for SR 141716A. Representative mutants were analyzed for agonist-induced inhibition of cyclic AMP accumulation, and the results indicated that G-protein coupling remained intact. Alanine substitution mutants were made to address whether it was the perturbation of the overall structure of the region or the displacement of particular side chains that was responsible for the loss of CP 55,940 binding. We conclude that a structurally intact EC1, but not the comparably short EC3, is essential for high-affinity CP 55,940 binding.