Structure of the VHS domain of human Tom1 (target of myb 1): insights into interactions with proteins and membranes

VHS domains are found at the N-termini of select proteins involved in intracellular membrane trafficking. We have determined the crystal structure of the VHS domain of the human Tom1 (target of myb 1) protein to 1.5 A resolution. The domain consists of eight helices arranged in a superhelix. The surface of the domain has two main features: (1) a basic patch on one side due to several conserved positively charged residues on helix 3 and (2) a negatively charged ridge on the opposite side, formed by residues on helix 2. We compare our structure to the recently obtained structure of tandem VHS-FYVE domains from Hrs [Mao, Y., Nickitenko, A., Duan, X., Lloyd, T. E., Wu, M. N., Bellen, H., and Quiocho, F. A. (2000) Cell 100, 447-456]. Key features of the interaction surface between the FYVE and VHS domains of Hrs, involving helices 2 and 4 of the VHS domain, are conserved in the VHS domain of Tom1, even though Tom1 does not have a FYVE domain. We also compare the structures of the VHS domains of Tom1 and Hrs to the recently obtained structure of the ENTH domain of epsin-1 [Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P., and Brünger, A. T. (2000) J. Cell Biol. 149, 537-546]. Comparison of the two VHS domains and the ENTH domain reveals a conserved surface, composed of helices 2 and 4, that is utilized for protein-protein interactions. In addition, VHS domain-containing proteins are often localized to membranes. We suggest that the conserved positively charged surface of helix 3 in VHS and ENTH domains plays a role in membrane binding.     

Determination of the secondary structure and folding topology of human interleukin-4 using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure of human recombinant interleukin-4 (IL-4) has been investigated by three-dimensional (3D) 15N- and 13C-edited nuclear Overhauser (NOE) spectroscopy on the basis of the 1H, 15N, and 13C assignments presented in the preceding paper [Powers, R., Garrett, D. S., March, C. J., Frieden, E. A., Gronenborn, A. M., & Clore, G. M. (1992) Biochemistry (preceding paper in this issue)]. Based on the NOE data involving the NH, C alpha H, and C beta H protons, as well as 3JHN alpha coupling constant, amide exchange, and 13C alpha and 13C beta secondary chemical shift data, it is shown that IL-4 consists of four long helices (residues 9-21, 45-64, 74-96, and 113-129), two small helical turns (residues 27-29 and 67-70), and a mini antiparallel beta-sheet (residues 32-34 and 110-112). In addition, the topological arrangement of the helices and the global fold could be readily deduced from a number of long-range interhelical NOEs identified in the 3D 13C-edited NOE spectrum in combination with the spatial restrictions imposed by three disulfide bridges. These data indicate that the helices of interleukin-4 are arranged in a left-handed four-helix bundle with two overhand connections.     

Dynamics and hydration of the alpha-helices of apolipophorin III

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein whose structure is represented as a bundle of five amphipathic alpha-helices. In order to study the properties of the helical domains of apolipophorin III, we designed and obtained five single-tryptophan mutants of Locusta migratoria apoLp-III. The proteins were studied by UV absorption spectroscopy, time-resolved and steady-state fluorescence spectroscopy, and circular dichroism. Fluorescence anisotropy, near-UV CD and solute fluorescence quenching studies indicate that the Trp residues in helices 1 (N-terminal) and 5 (C-terminal) have the highest conformational flexibility. These two residues also showed the highest degree of hydration. Trp residues in helices 3 and 4 display the lowest mobility, as assessed by fluorescence anisotropy and near UV CD. The Trp residue in helix 2 is protected from the solvent but shows high mobility. As inferred from the properties of the Trp residues, helices 1 and 5 appear to have the highest conformational flexibility. Helix 2 has an intermediate mobility, whereas helices 3 and 4 appear to constitute a highly ordered domain. From the configuration of the helices in the tertiary structure of the protein, we estimated the relative strength of the five interhelical interactions of apoLp-III. These interactions can be ordered according to their apparent stabilizing strengths as: helix 3-helix 4 > helix 2-helix 3 > helix 4-helix 1 approximately helix 2-helix 5 > helix 1-helix 5. A new model for the conformational change that is expected to occur upon binding of the apolipoprotein to lipid is proposed. This model is significantly different from the currently accepted model (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesemberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, M. (1991) Biochemistry 30, 603-608). The model presented here predicts that the relaxation of the tertiary structure and the concomitant exposure of the hydrophobic core take place through the disruption of the weak interhelical contacts between helices 1 and 5. To some extent, the weakness of the helix 1-helix 5 interaction would be due to the parallel arrangement of these helices.     

Structure of the detergent phase and protein-detergent interactions in crystals of the wild-type (strain Y) Rhodobacter sphaeroides photochemical reaction center

Rhodobacter sphaeroides (strain Y) reaction center (RC) crystals were grown in the presence of n-octyl beta-glucoside (beta-OG). In order to determine the structure of the detergent phase in these crystals, low-resolution neutron diffraction experiments were performed at different contrasts obtained by varying the H2O/D2O ratio in the solvent. From the contrast variation data and from the RC atomic coordinates determined by X-ray diffraction [Arnoux, B., Ducruix, A., Reiss-Husson, F., Lutz, M., Norris, J., Schiffer, M., & Chang, C. H. (1989) FEBS Lett. 258, 47-50], a model was obtained for the structure of the detergent phase in the crystal. The detergent forms a ring-shaped micelle surrounding the most hydrophobic part of the transmembrane alpha helices of the RC. Each detergent ring is connected to two next-neighbor rings by intermicellar bridges. The detergent phase is organized thus in infinite zigzag chains parallel to the b axis of the P2(1)2(1)2(1) unit cell. The main interactions between beta-OG molecules and the RC molecules are hydrophobic and are localized at the level of the transmembrane alpha helices. This interaction replaces the phospholipid-protein interaction existing in vivo in the membrane and, to some extent, also the light harvesting complex-protein interaction. Secondary hydrophilic interactions are found between a few of the charged residues of the H subunit and the hydrophilic surface of the detergent ring from a neighboring RC molecule. A comparison with a previous study on Rhodopseudomonas viridis crystals [which grow in the presence of lauryldimethylamine N-oxide (LDAO) and belong to a different space group] [Roth, M., Lewit-Bentley, A., Michel, H., Deisenhofer, J., Huber, R., & Oesterhelt, D. (1989) Nature 340, 659-661] shows a quasi identity of shape and position of the beta-OG and LDAO rings around the transmembrane alpha helices. The secondary interactions, involving in both cases the external surface of the H subunit, differ because of the different molecular packing in the two space groups. The role and structural requirements of the detergent in the crystallization process are discussed.     

Mapping of the leptin binding sites and design of a leptin antagonist

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.     

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.     

Two distinct functional sites of human interleukin 4 are identified by variants impaired in either receptor binding or receptor activation.

Interleukin 4 (IL-4) exerts a decisive role in the coordination of protective immune responses against parasites, particularly helminths. A disregulation of IL-4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human IL-4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4-helix-bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL-4 variants deficient in binding to the extracellular domain of the IL-4 receptor (IL-4R(ex)). In parallel, up to 1000-fold increased concentrations of this type of variant were required to induce T-cell proliferation and B-cell CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild-type). IL-4 variants affected at site 2 exhibited partial agonist activity during T-cell proliferation; however, they still bound with high affinity to IL-4R(ex). [The generation of an IL-4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO J., 11, 3237-3244)]. These findings indicate that IL-4 functions by binding IL-4R(ex) via site 1 which is constituted by residues on helices A and C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A model of the complex between interleukin-4 and its receptors

A three-dimensional model of interleukin-4 (IL-4) bound to one molecule each of the high- and low-affinity receptors (IL-4R and IL-2Rγ) was built, using the crystal structure of the complex of human growth hormone (HGH) with its receptor (HGHR) as a starting model. The modeling of IL-4 with its receptors was based on the conservation of the sequences and on the predicted structural organization for cytokine receptors, and assuming that the binding mode of the ligands would be similar. Analysis of the interface between IL-4 and both receptor molecules was carried out to reveal which residues are important for complex formation. The modeling procedures showed that there were no major problems in maintaining a reasonable fit of IL-4 with the two receptor molecules, in a manner analogous to the complex of HGH–HGHR. Many of the residues that appear by modeling to be important for binding between IL-4 and the receptors have been previously implicated in that role by different methods. A striking motif of aromatic and positively charged residues on the surface of the C-terminal domains of the receptors is highly conserved in the structure of HGH–HGHR and in the models of IL-4 complexed with its receptors. © 1995 Wiley-Liss, Inc.     

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.     

Solution structure of peptide toxins that block mechanosensitive ion channels

Mechanosensitive channels (MSCs) play key roles in sensory processing and have been implicated as primary transducers for a variety of cellular responses ranging from osmosensing to gene expression. This paper presents the first structures of any kind known to interact specifically with MSCs. GsMTx-4 and GsMtx-2 are inhibitor cysteine knot peptides isolated from venom of the tarantula, Grammostola spatulata (Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., Baumgarten, C. M., and Sachs, F. (2000) J. Gen. Physiol. 115, 583-598). Inhibition of cationic MSCs by the higher affinity GsMtx-4 (K(D) approximately 500 nm) reduced cell size in swollen and hypertrophic heart cells, swelling-activated currents in astrocytes, and stretch-induced arrhythmias in the heart. Despite the relatively low affinity, no cross-reactivity has been found with other channels. Using two-dimensional NMR spectroscopy, we determined the solution structure of GsMTx-4 and a lower affinity (GsMTx-2; K(D) approximately 6 microm) peptide from the same venom. The dominant feature of the two structures is a hydrophobic patch, utilizing most of the aromatic residues and surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 and GsMTx-2 may underlie the selectivity of these peptides.     

Multiple replacements at position 211 in the alpha subunit of tryptophan synthase as a probe of the folding unit association reaction

Equilibrium and kinetic studies on the folding of a series of amino acid replacements at position 211 in the alpha subunit of tryptophan synthase from Escherichia coli were performed in order to determine the role of this position in the rate-limiting step in folding. Previous studies [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965-2974] have shown that the rate-limiting step corresponds to the association/dissociation of the amino (residues 1-188) and carboxy (residues 189-268) folding units. In terms of the secondary structure, the amino folding unit consists of the first six strands and five alpha helices of this alpha/beta barrel protein. The carboxy folding unit comprises the remaining two strands and three alpha helices; position 211 is in strand 7. Replacement of the wild-type glycine at position 211 with serine, valine, and tryptophan at most alters the rate of dissociation of the folding units; association is not changed significantly. In contrast, glutamic acid and arginine dramatically decelerate and accelerate, respectively, both association and dissociation. The difference in effects is attributed to long-range electrostatic interactions for these charged side chains; steric effects and/or hydrogen bonding play lesser roles. When considered with previous data on replacements at other positions in the alpha subunit [Hurle, M. R., Tweedy, N. B., & Matthews, C. R. (1986) Biochemistry 25, 6356-6360], it is clear that beta strands 6 (in the amino folding unit) and 7 (in the carboxy folding unit and containing position 211) dock late in the folding process.     

Structural model of human IL-13 defines the spatial interactions with the IL-13Ralpha/IL-4Ralpha receptor

Interleukin-13 (IL-13) plays a key role in immune responses and inflammation. A structural model of human IL-13 (HuIL-13) based on the nuclear magnetic resonance and X-ray structure of IL-4 is put forward. Unlike previous models, this model is based on new sequence alignments that take into account the formation of the two disulfide linkages that have been determined experimentally. The proposed structure of human IL-13 is similar to IL-4, consisting of a four helix bundle with hydrophobic residues lining the core of the molecule and surface polar residues showing a high degree of solvent accessibility. Regions of HuIL-13 that are critical for the interaction with its receptors are explored and discussed in relation to existing mutagenic studies. From these studies we predict that helices A and C of HuIL-13 interact with the IL-4 receptor alpha (IL-4Ralpha) region and helix D is responsible for the interaction with the IL-13 receptor alpha 1 (IL-13Ralpha1) receptor.     

A Unique Loop Structure in Oncostatin M Determines Binding Affinity toward Oncostatin M Receptor and Leukemia Inhibitory Factor Receptor

Oncostatin M (OSM) and leukemia inhibitory factor are pleiotropic cytokines that belong to the interleukin-6 (IL-6) family. These cytokines play a crucial role in diverse biological events like inflammation, neuroprotection, hematopoiesis, metabolism, and development. The family is grouped together based on structural similarities and their ability to activate the transmembrane receptor glycoprotein 130 (gp130). The common structure among these cytokines defines the spacing and the orientation of binding sites for cell surface receptors. OSM is unique in this family as it can signal using heterodimers of gp130 with either leukemia inhibitory factor receptor (LIFR) (type I) or oncostatin M receptor (OSMR) (type II). We have identified a unique helical loop on OSM between its B and C helices that is not found on other IL-6 family cytokines. This loop is located near the "FXXK" motif in active site III, which is essential for OSM's binding to both LIFR and OSMR. In this study, we show that the BC loop does not play a role in OSM's unique ability to bind OSMR. Shortening of the loop enhanced OSM's interaction with OSMR and LIFR as shown by kinetic and equilibrium binding analysis, suggesting the loop may hinder receptor interactions. As a consequence of improved binding, these structurally modified OSMs exhibited enhanced biological activity, including suppressed proliferation of A375 melanoma cells.     

NMR solution structure of ImB2, a protein conferring immunity to antimicrobial activity of the type IIa bacteriocin, carnobacteriocin B2

Bacteriocins produced by lactic acid bacteria are potent antimicrobial compounds which are active against closely related bacteria. Producer strains are protected against the effects of their cognate bacteriocins by immunity proteins that are located on the same genetic locus and are coexpressed with the gene encoding the bacteriocin. Several structures are available for class IIa bacteriocins; however, to date, no structures are available for the corresponding immunity proteins. We report here the NMR solution structure of the 111-amino acid immunity protein for carnobacteriocin B2 (ImB2). ImB2 folds into a globular domain in aqueous solution which contains an antiparallel four-helix bundle. Extensive packing by hydrophobic side chains in adjacent helices forms the core of the protein. The C-terminus, containing a fifth helix and an extended strand, is held against the four-helix bundle by hydrophobic interactions with helices 3 and 4. Most of the charged and polar residues in the protein face the solvent. Helix 3 is well-defined to residue 55, and a stretch of nascent helix followed by an unstructured loop joins it to helix 4. No interaction is observed between ImB2 and either carnobacteriocin B2 (CbnB2) or its precursor. Protection from the action of CbnB2 is only observed when ImB2 is expressed within the cell. The loop between helices 3 and 4, and a hydrophobic pocket which it partially masks, may be important for interaction with membrane receptors responsible for sensitivity to class IIa bacteriocins.     

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.     

Ca2+ -ATPase structure in the E1 and E2 conformations: mechanism,helix-helix and helix-lipid interactions

The determination of the crystal structure of the Ca(2+)-ATPase of sarcoplasmic reticulum (SR) in its Ca(2+)-bound [Nature 405 (2000) 647] and Ca(2+)-free forms [Nature 418 (2002) 605] gives the opportunity for an analysis of conformational changes on the Ca(2+)-ATPase and of helix-helix and helix-lipid interactions in the transmembrane (TM) region of the ATPase. The locations of the ends of the TM alpha-helices on the cytoplasmic side of the membrane are reasonably well defined by the location of Trp residues and by the location of Lys-262 that snorkels up to the surface. The locations of the lumenal ends of the helices are less clear. The position of Lys-972 on the lumenal side of helix M9 suggests that the hydrophobic thickness of the protein is only about 21 A, rather than the normal 30 A. The experimentally determined TM alpha-helices do not agree well with those predicted theoretically. Charged headgroups are required for strong interaction of lipids with the ATPase, consistent with the large number of charged residues located close to the lipid-water interface. Helix packing appears to be rather irregular. Packing of helices M8 and M10 is of the 3-4 ridges-into-grooves or knobs-into-holes types. Packing of helices M5 and M7 involves two Gly residues in M7 and one Gly residue in M5. Packing of the other helices generally involves just one or two residues on each helix at the crossing point. The irregular packing of the TM alpha-helices in the Ca(2+)-ATPase, combined with the diffuse structure of the ATPase on the lumenal side of the membrane, is suggested to lead to a relative low activation energy for changing the packing of the TM alpha-helices, with changes in TM alpha-helical packing being important in the process of transfer of Ca(2+) ions across the membrane. The inhibitor thapsigargin binds in a cleft between TM alpha-helices M3, M5 and M7. It is suggested that this and other similar clefts provide binding sites for a variety of hydrophobic molecules affecting the activity of the Ca(2+)-ATPase.     

Mucin biosynthesis: upregulation of core 2 beta 1,6 N-acetylglucosaminyltransferase by retinoic acid and Th2 cytokines in a human airway epithelial cell line

Vitamin A and the T helper 2 cytokines IL-4 and IL-13 play important roles in the induction of mucin gene expression and mucus hypersecretion. However, the effects of these agents on enzymes responsible for mucin glycosylation have received little attention. Here, we report the upregulation of core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity both by all-trans retinoic acid (RA) and by IL-4 and IL-13 in the H292 airway epithelial cell line. Northern blotting analysis showed that the M isoform of C2GnT, which is expressed in mucus-secreting tissues and can form all mucin glycan beta1,6-branched structures, including core 2, core 4, and blood group I antigen, was upregulated by both RA and IL-4/13. The L isoform, which forms only the core 2 structure, was moderately upregulated by IL-4/13 but not by RA. Enhancement of the M isoform of C2GnT by RA was abolished by an inhibitor of RA receptor alpha, implicating RA receptor alpha in the effect of RA. Likewise, an inhibitor of the Janus kinase 3 pathway blocked the enhancing effects of IL-4/13 on the L and M isoforms of C2GnT, suggesting a role of this pathway in the upregulation of these two C2GnTs by these cytokines. Taken together, the results suggest that IL-4/13 T helper 2 cytokines and RA can alter the activity of enzymes that synthesize branching mucin carbohydrate structure in airway epithelial cells, potentially leading to altered mucin carbohydrate structure and properties.     

Comprehensive analysis of the numbers,lengths and amino acid compositions of transmembrane helices in prokaryotic,eukaryotic and viral integral membrane proteins of high-resolution structure

We report a comprehensive analysis of the numbers, lengths and amino acid compositions of transmembrane helices in 235 high-resolution structures of integral membrane proteins. The properties of 1551 transmembrane helices in the structures were compared with those obtained by analysis of the same amino acid sequences using topology prediction tools. Explanations for the 81 (5.2%) missing or additional transmembrane helices in the prediction results were identified. Main reasons for missing transmembrane helices were mis-identification of N-terminal signal peptides, breaks in α-helix conformation or charged residues in the middle of transmembrane helices and transmembrane helices with unusual amino acid composition. The main reason for additional transmembrane helices was mis-identification of amphipathic helices, extramembrane helices or hairpin re-entrant loops. Transmembrane helix length had an overall median of 24 residues and an average of 24.9 ± 7.0 residues and the most common length was 23 residues. The overall content of residues in transmembrane helices as a percentage of the full proteins had a median of 56.8% and an average of 55.7 ± 16.0%. Amino acid composition was analysed for the full proteins, transmembrane helices and extramembrane regions. Individual proteins or types of proteins with transmembrane helices containing extremes in contents of individual amino acids or combinations of amino acids with similar physicochemical properties were identified and linked to structure and/or function. In addition to overall median and average values, all results were analysed for proteins originating from different types of organism (prokaryotic, eukaryotic, viral) and for subgroups of receptors, channels, transporters and others.     

Disulfide bond assignment and identification of regions required for functional activity of oncostatin M

Oncostatin M is a polypeptide cytokine having unique structure and diverse biological activities, including the ability to inhibit growth of certain cultured tumor cells. Here we have determined the disulfide bonding pattern of recombinant oncostatin M and have used site-directed mutagenesis to identify regions of this molecule necessary for receptor binding and growth inhibitory activities. Two intramolecular disulfide bonds, C6-C127 and C49-C167, were identified in recombinant oncostatin M. Analysis of mutations at each of the five cysteines in oncostatin M indicated that mutants C49S and C167S were inactive (less than 1/10 wild type activity) in growth inhibitory assays and radioreceptor assays. Carboxyl-terminal deletion mutations terminating at S185 and beyond were active, but further shortening abolished activity in both assays. Two deletion mutants proximal to C49 (delta 22-36 and delta 44-47) and insertion mutant GAG77 also were inactive. One deletion mutant, delta 87-90, had significantly (approximately 3-fold) increased activities in both growth inhibitory assays and radioreceptor assays. A potential amphiphilic domain was identified beginning at C167 and extending toward the carboxyl terminus. Two mutants having altered hydrophobic residues within this domain (F176G and F184G) were inactive, suggesting that these residues are required for proper conformation of the receptor binding site. Taken together, these results indicate that biological activity of oncostatin M requires discontinuous regions of the molecule, including residues near the essential disulfide bond, C49-C167, and within a putative amphiphilic helix at the carboxyl terminus. Oncostatin M thus belongs to a growing family of cytokines whose interactions with their respective receptors are mediated in part by known or predicted carboxyl-terminal amphiphilic helices.     

Three-dimensional solution structure of a unique S100 protein

S100A13 is a homodimeric protein that belongs to the S100 subfamily of EF-hand Ca2+-binding proteins. S100A13 exhibits unique physical and functional properties not observed in other members of the S100 family. S100A13 is crucial for the non-classical export of acidic fibroblast growth factors (FGFs-1), which lack signal peptide at their N-terminal end. In the present study, we report the three-dimensional solution structure of Ca2+-bound S100A13 using a variety of 3D NMR experiments. The structure of S100A13 is globular with four helices and an antiparallel beta-sheet in each subunit. The dimer interface is formed mainly by an antiparallel arrangement of helices H1, H1', H4, and H4'. Isothermal titration calorimetry (ITC) experiments show that S100A13 binds non-cooperatively to four calcium ions. Prominent differences exist between the three-dimensional structures of S100A13 and other S100 proteins. The hydrophobic pocket that largely contributes to protein-protein interactions in other S100 proteins is absent in S100A13. The structure of S100A13 is characterized by a large patch of negatively charged residues flanked by dense cationic clusters contributed largely by the positively charged residues located at the C-terminal end. Results of ITC experiments reveal that S100A13 lacking the C-terminal segment (residues 88-98) fails to bind FGF-1. The three-dimensional structure of S100A13 not only provides useful clues on its role in the non-classical export of signal peptide-less proteins such as FGF-1 but also paves the way for rational design of drugs against FGF-induced tumors.