Contributions of Phenylalanine 335 to Ligand Recognition by Human Surfactant Protein D (2006)

Surfactant protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Interactions of SP-D with microorganisms and organic antigens involve binding of glycoconjugates to the C-type lectin carbohydrate recognition domain (CRD). A trimeric fusion protein encoding the human neck+CRD bound to the aromatic glycoside p-nitrophenyl-α-d-maltoside with nearly a log-fold higher affinity than maltose, the prototypical competitor. Maltotriose, which has the same linkage pattern as the maltoside, bound with intermediate affinity. Site-directed substitution of leucine for phenylalanine 335 (Phe-335) decreased affinities for the maltoside and maltotriose without significantly altering the affinity for maltose or glucose, and substitution of tyrosine or tryptophan for leucine restored preferential binding to maltotriose and the maltoside. A mutant with alanine at this position failed to bind to mannan or maltose-substituted solid supports. Crystallographic analysis of the human neck+CRD complexed with maltotriose or p-nitrophenyl-maltoside showed stacking of the terminal glucose or nitrophenyl ring with the aromatic ring of Phe-335. Our studies indicate that Phe-335, which is evolutionarily conserved in all known SP-Ds, plays important, if not critical, roles in SP-D function. Surfactant protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Interactions of SP-D with microorganisms and organic antigens involve binding of glycoconjugates to the C-type lectin carbohydrate recognition domain (CRD). A trimeric fusion protein encoding the human neck+CRD bound to the aromatic glycoside p-nitrophenyl-α-d-maltoside with nearly a log-fold higher affinity than maltose, the prototypical competitor. Maltotriose, which has the same linkage pattern as the maltoside, bound with intermediate affinity. Site-directed substitution of leucine for phenylalanine 335 (Phe-335) decreased affinities for the maltoside and maltotriose without significantly altering the affinity for maltose or glucose, and substitution of tyrosine or tryptophan for leucine restored preferential binding to maltotriose and the maltoside. A mutant with alanine at this position failed to bind to mannan or maltose-substituted solid supports. Crystallographic analysis of the human neck+CRD complexed with maltotriose or p-nitrophenyl-maltoside showed stacking of the terminal glucose or nitrophenyl ring with the aromatic ring of Phe-335. Our studies indicate that Phe-335, which is evolutionarily conserved in all known SP-Ds, plays important, if not critical, roles in SP-D function. Surfactant Protein D (SP-D) 2The abbreviations used are: SP-D, surfactant protein D; CRD, carbohydrate recognition domain; Glc, glucose; NCRD, human neck+CRD; NP, nitrophenyl; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone. is a collagenous C-type lectin (collectin) that contributes to antimicrobial host defense and immune regulation in the lung and certain extrapulmonary tissues (1Clark H. Reid K.B. Immunobiology. 2002; 205: 619-631Crossref PubMed Scopus (38) Google Scholar, 2Crouch E.C. Whitsett J.A. Innate Immunity. 2002; (Humana Press Inc., Rotowa, NJ): 205-230Crossref Google Scholar, 3Hawgood S. Poulain F.R. Annu. Rev. Physiol. 2001; 63: 495-519Crossref PubMed Scopus (125) Google Scholar, 4Crouch E. Wright J.R. Annu. Rev. Physiol. 2001; 63: 521-554Crossref PubMed Scopus (570) Google Scholar). Like many other effectors of innate immunity, SP-D is a pattern recognition molecule with a variety of potential ligands, including microorganisms, organic particulate antigens, and apoptotic cells. However, there is also evidence that SP-D contributes to pulmonary surfactant homeostasis, in part by regulating the organization and epithelial uptake of surfactant lipids (5Ikegami M. Na C.L. Korfhagen T.R. Whitsett J.A. Am. J. Physiol. 2005; 288: L552-L561Crossref PubMed Scopus (79) Google Scholar). SP-D is a member of a family of collectins, or collagenous C-type lectins (6Holmskov U.L. APMIS. 2000; 100: 1-59Google Scholar). In mammals, this family also includes surfactant protein A (SP-A) and serum mannose binding lectin. Like most other collectins, SP-D is assembled as multimeric complexes of trimeric subunits, or as trimers (7Crouch E.C. Respir. Res. 2000; 1: 93-108Crossref PubMed Scopus (167) Google Scholar). Each trimer includes an N-terminal cross-linking and collagen domain; a trimeric neck domain; and a C-terminal trimeric array of C-type carbohydrate recognition domains (CRDs). Crystallographic studies of trimeric human SP-D neck+CRD domains have shown that maltose, a preferred saccharide ligand, binds to calcium via the vicinal 3- and 4-OH groups of the non-reducing glucose, previously designated calcium ion 1 and glucose 1 (Glc1), respectively (8Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Crossref PubMed Scopus (97) Google Scholar). These interactions are further stabilized by hydrogen bonding of Glc1 to amino acid side chains that also coordinate with calcium ion 1. The present studies were prompted by the initial observation that p-nitrophenyl-α-d-maltoside (p-NP-maltoside) is a potent inhibitor of SP-D binding to mannan. We hypothesized that there were interactions of the aromatic substituent with a hydrophobic or aromatic group in proximity to calcium ion 1. Preliminary modeling focused our attention on phenylalanine 335 (Phe-335) (Fig. 1A). To test our hypothesis, we used a functional, trimeric neck+CRD (NCRD) fusion protein containing the neck+CRD domains of human SP-D (Fig. 1, B–D) (9Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and introduced site-directed substitutions at position 335. We also examined the crystal structures of purified human trimeric neck+CRD complexed with the p-nitrophenyl-maltoside and maltotriose. The p-nitrophenyl-α-d-maltoside (number 487542) was from EMD Biosciences (La Jolla, CA). The p-nitrophenyl-β-d-maltoside (N1884), p-nitrophenyl-α-d-glucopyranoside (N1377), p-nitrophenyl-α-d-galactopyranoside (N0877), maltotriose (M8378), and maltotetraose (Dp4, Supelco) were from Sigma-Aldrich. Trypsin-TPCK (T-8802) and all simple sugars were also from Sigma. Expression, Purification, and Biochemical Characterization of Trimeric Neck+CRDs—The design, expression, and characterization of N-terminal-tagged, trimeric human NCRD fusion proteins has been previously described (9Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). For these studies, we performed site-directed substitutions at position 335 within the CRD, as defined by numbering of the full-length protein: alanine (F335A), leucine (F335L), tyrosine (F335Y), and tryptophan (F335W) (Fig. 1, A–C). DNA sequences were verified by automated sequencing of the entire coding sequence of the fusion protein. Competent cells were transformed with wild-type or mutant constructs in pET-30a(+) vector, and expressed proteins were isolated from inclusion bodies (9Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). After refolding and oligomerization, the fusion proteins were purified by nickel affinity chromatography and trimers were isolated by gel filtration (9Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Protein concentrations were determined using the bicinchoninic acid assay with bovine serum albumin as standard. All recombinant proteins isolated from inclusion bodies had <10 pg of endotoxin/μg of purified protein. The binding of trimeric NCRDs to yeast mannan was examined as previously described (9Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). For competition assays, proteins were added to wells in the presence of various concentrations of competing inhibitors. The concentration of competitor (mm) required for 50% inhibition of binding (I50) was calculated. Protease protection assays of F335A were performed using trypsin-TPCK and modifications of published procedures (10Mullin N.P. Hall K.T. Taylor M.E. J. Biol. Chem. 1994; 269: 28405-28413Abstract Full Text PDF PubMed Google Scholar). Following digestion in the absence and presence of various concentrations of calcium, the NCRDs were resolved by SDS-PAGE, visualized by staining, and quantified by densitometry. The percent of cleavage relative to controls incubated in the absence of enzyme was plotted versus the final calcium concentration, and the calcium concentration required for 50% protection (Ca50) was calculated. Purification of the Human NCRD for Crystallographic Studies—The conditions of isolation were modified to decrease the final ionic strength, eliminate Tris, and increase the final protein concentration. Refolded proteins were dialyzed versus 500 mm NaCl, 10 mm Hepes, pH 7.5 (HBS) containing 10 mm imidazole and applied to a nickel affinity column (9Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The fusion protein was eluted with an imidazole gradient and dialyzed versus 150 mm NaCl, 10 mm Hepes, pH 7.5, containing 10 mm calcium chloride (HBSC). The trimeric neck+CRDs were then liberated by incubation with enterokinase (0.0015%, w/w) for up to 20 h at 37 °C. Following adsorption of the enzyme, the proteins were concentrated to 8 mg/ml using an Amicon Ultra (MWCO 10,000; Millipore) and applied to a Superose 12 column equilibrated with HBSC. The pooled trimer fractions were reconcentrated to ∼15 mg/ml. The protein showed a single peak with a polydispersity of <15% by dynamic light scattering. Crystallographic Analysis of Ligand Complexes—Crystals of native human NCRD were grown by the hanging droplet vapor diffusion method, mixing 2 μl of protein at 15 mg/ml in 10 mm Hepes, pH 7.4, 150 mm NaCl, 10 mm CaCl2 with 2 μl of 12% polyethylene glycol 8000, 100 mm Hepes, pH 7.5, and equilibrating over 1 ml of 12% polyethylene glycol 8000, 100 mm Hepes, pH 7.5. Crystals with dimensions of ∼0.2–0.3 mm on a side grew within a week. Ligands were soaked into crystals by dissolving ligand in mother liquor and transferring a crystal into the resulting solution for a period of 1–2 h. The p-nitrophenyl maltoside was soaked at a concentration of 50 mm and maltotriose at a concentration of 250 mm. After soaking, the crystal was flash frozen in a 100°K stream of nitrogen gas at the beamline. All data were collected at beamline X8C at the National Synchrotron Light Source, Brookhaven National Laboratory. Data were processed using DENZO and SCALEPACK (11Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Data collection statistics are shown under “Results,” Table 2.TABLE 2Data collection and refinement statisticsData setp-Nitrophenol-α -d-maltosideMaltotrioseSpace groupP21P21Cell dimensionsa = 55.43, b = 106.4, c = 55.08, beta = 92.65a = 55.73, b = 108.93, c = 55.84, beta = 91.13Wavelength (Å)1.11.1Resolution range (Å)50-1.950-1.85Measured reflections805322630158Unique reflections5092956776Completeness (%) overall (final shell)99.8 (99.8)99.9 (99.8)〈I/σI 〉 (final shell)18.8 (5.6)20.3 (4.8)Rmerge(%) (final shell)0.07 (0.28)0.05 (0.30)Refinement Rcryst0.2140.221 Rfree0.2380.247Atoms/Asymmetric unit Protein34623462 Water259277 Ions99 Ligand96102 Total38263850Average B (Å2) Main chain27.722.7 Side chain29.925.2 Ligand36.232.0 All atoms29.324.5R.m.s.d.aRoot mean square deviation. Bonds (Å)0.0060.006 Angles (°)1.1371.105a Root mean square deviation. Open table in a new tab Models of bound saccharide were built based on electron density maps derived using diffraction data and phases of the protein component of the structure of SP-D determined in the presence of maltose, Protein Data Bank accession code 1PWB. (8Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Crossref PubMed Scopus (97) Google Scholar). Difference maps showed clear density for the bound ligands. Topology and parameter files for refinement of models of maltotriose and the maltoside were determined using the program Xplo2d (12Kleywegt G.J. CCP4/ESF-EACBM Newsletter on Protein Crystallography. 1995; 31: 45-50Google Scholar). Refinement was performed using CNS (13Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) and model building and rebuilding in O (14Jones T.A. Kjeldgaard M. O-The Manual. 1992; (Uppsala Software Factory, Uppsala, Sweden)Google Scholar). Refinement statistics are shown in Table 2. Figures were prepared using Swiss-PDBView (15Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus Google and E.C. J. Chem. PubMed Scopus Google Scholar). Human NCRDs the of an or interactions are known to to the interactions of various including C-type lectins M.E. K. Methods Enzymol. 2003; PubMed Scopus Google Scholar). of and lectins have with hydrophobic as aromatic H. H. H. N. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. B. M. J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). maltose is the most potent simple competitor of SP-D binding to various ligands, we examined the binding of human SP-D NCRDs to The aromatic maltoside was a potent inhibitor of human NCRD binding to nearly a log-fold potent than the prototypical SP-D maltose, an glucose (Fig. Table of human NCRD by and aromatic NCRD of Open table in a new tab To and the of we examined other p-nitrophenyl The of the substituent were for the glycoside and with known saccharide of The of was with glucose and was than a competitor not The was potent than the maltoside (Fig. binding of aromatic is by the and of the p-nitrophenyl group on binding affinity on the of the aromatic Human NCRDs to these we examined binding of the human NCRD to an glucose with a of a ring and the same structure as the maltoside. was potent than maltose as a competitor of NCRD binding to potent than the (Fig. Table For these the of of simple in competition assays using solid mannan (Fig. Table 1, data not The of glucose (Glc1), maltose and maltotriose ligand interactions and However, there was further increase in binding affinity of a was potent than maltose, than maltotriose for Site-directed of with published data of human SP-D NCRD complexed with maltose (8Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Crossref PubMed Scopus (97) Google Scholar). visualized in that the the binding to the binding of a trimeric CRD, with from the binding our a binding for Glc1 of maltotriose on the terminal sugars of the bound of these the of other in the of the carbohydrate binding other of proteins aromatic to or carbohydrate our attention focused on of the a that is and the calcium ion at the carbohydrate binding Site-directed of for to lectins many at the position to Phe-335, including alanine 2003; PubMed Scopus (97) Google Scholar). we that a substitution of alanine for to the of the aromatic side The mutant was isolated in from inclusion and the were from the wild-type protein. In the purified protein on with the wild-type human NCRD in the absence and presence of with the of (Fig. and data not of the alanine mutant showed with mannan in solid binding assays the protein not bind to serum albumin or to using conditions of saccharide affinity chromatography not the ring of to with the side of a conserved of the C-type lectin that with calcium ion 1, we potential of the on calcium binding affinity. However, saccharide binding was not by calcium concentrations as as 50 mm not a that has been used to binding of C-type lectin with calcium binding affinity K. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). In there was in the of the wild-type protein and F335A to cleavage in the presence or absence of calcium (10Mullin N.P. Hall K.T. Taylor M.E. J. Biol. Chem. 1994; 269: 28405-28413Abstract Full Text PDF PubMed Google Scholar, K. 1992; PubMed Scopus Google Scholar). The wild-type and mutant neck+CRD domains were to cleavage by w/w) for of up to 2 h at in the presence of concentrations of However, proteins were in the absence of calcium, and the calcium concentration with 50% (Ca50) was for F335A than the wild-type mm = 2 versus mm = These are to the calcium concentration required for binding of the wild-type fusion protein to mannan (9Crouch E. Tu Y. Briner D. McDonald B. Smith K. Holmskov U. Hartshorn K. J. Biol. Chem. 2005; 280: 17046-17056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). of at 335 the for and performed a substitution by with a hydrophobic amino The purified protein from the wild-type protein and F335A on (Fig. However, in with showed binding to to the wild-type protein (Fig. The for the mutant was = as with = for the wild-type In binding was by the including maltose (Fig. However, the of maltotriose and p-nitrophenyl-maltoside were as with the with affinities to glucose and The binding affinities for glucose, maltose, and other sugars were or decreased as with the wild-type protein (Fig. data not were for of mutant protein. or for to that was as a lectin showed preferential with maltotriose or we hypothesized that the ring structure is for interactions with these ligands. we tyrosine or tryptophan for Phe-335, using the DNA as a were isolated as trimers and were from the wild-type fusion protein on (Fig. or showed binding to mannan not binding of was with and the wild-type binding of was in with a of and also the wild-type protein in relative for maltotriose over maltose (Fig. the of maltotriose was for than for the wild-type protein or other were of the on the of other examined saccharide of the Human NCRD with and further the binding the crystal structure of the human trimeric NCRD was determined as complexes with maltotriose and p-nitrophenyl-α-d-maltoside and Table shown in of maltotriose is over with to the ring of Phe-335, that is nearly over the of the phenylalanine by a of the of the ring is relative to Glc1 (Fig. The same binding was for Glc1 and on of bound and the nitrophenyl ring was with a over at a of (Fig. of a single with maltotriose. from to with and the binding at the same and in the same shown as a model with in that are within of the The of maltotriose are in of the wild-type human NCRD with and maltotriose. model and electron density at of maltotriose and of The of maltotriose are of the structures of the same of complexes of and maltotriose of maltose and maltotriose bound to the SP-D The protein model the maltotriose is shown as a model in and maltotriose as a model in The maltose was by of in this using the structure of Protein Data Bank accession 1PWB. of the hydrogen protein and maltotriose are shown by ring structures showed other interactions with the is a hydrogen from the side of to the of the as the A is and of the of the maltoside. hydrogen is in A and B from the side nitrogen of to the of of maltotriose and in all from the of to the of the maltoside. with the previously published maltose is Phe-335, and there is an of 1 (Fig. with that not Phe-335, there is also an of the with a of the aromatic ring not stacking of the carbohydrate ring of with the aromatic ring of to the overall binding of the and protein In the side of is to the of Glc1 and However, the of the side by hydrogen and other interactions and that the for hydrogen from to the or ring of the ligand is that the ligand is stabilized by the proximity of the groups to the on the The and data indicate that the aromatic ring of a that maltotriose as a competitor than the prototypical saccharide ligand, In the structure of the side at position 335 the solid interactions of SP-D with a is evolutionarily conserved all known SP-D and is calcium ion 1 and the carbohydrate binding (Fig. K. Reid K.B. Full Text Full Text PDF Scopus Google Scholar). this position is conserved C-type other aromatic or hydrophobic in this or ring interactions are known to a variety of interactions K. of Inc., PubMed Scopus Google and there is for this other C-type with to recognition N.P. Taylor M.E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the higher binding affinity for the maltotriose from the of the of aromatic as with stacking of the aromatic ring However, there are also the of hydrogen and These with Phe-335, an saccharide recognition at the ligand binding is also in and SP-D, is conserved in all known SP-Ds, a conserved binding the substitution of tryptophan for phenylalanine significantly the affinity for maltotriose as with the maltoside, as a of the of the showed a than maltotriose. We were to to the preferred solution of this However, the that interactions with binding to terminal sugars or structures in In the previously published maltose of maltose were visualized in a single (8Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Crossref PubMed Scopus (97) Google Scholar). However, in the present all were by maltotriose or the maltoside, and all of were to of the complexes by interactions or the nitrophenyl our are with the of (8Shrive A.K. Tharia H.A. Strong P. Kishore U. Burns I. Rizkallah P.J. Reid K.B. Greenhough T.J. J. Mol. Biol. 2003; 331: 509-523Crossref PubMed Scopus (97) Google Scholar) with to the and of Glc1 Our are also with the that which is on the N-terminal of the SP-D with of maltose K. Reid K.B. Full Text Full Text PDF Scopus Google Scholar). within the of maltotriose and the maltoside, is to with the of Glc1 a for the affinity of human SP-D for maltose as with However, the of these interactions on the of and and the side which from to showed binding to mannan and was bound to stacking interactions with an aromatic ring at 335 are not required for binding to solid ligands. the other showed binding to as a of hydrogen bonding In the with F335A that a hydrophobic is required at position 335 for binding to at solid we not in F335A or on characterization of the mutant is in The other lung surfactant protein A has a conserved tyrosine at the same position as Phe-335. is within a previously to a in ligand binding T.R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the aromatic of and in are to and the of the ring of these are by alanine in SP-D, the potential of the of the aromatic In our studies that in an carbohydrate binding at the ligand binding We that the aromatic ring binding of the by stacking with carbohydrate or hydrophobic in the same or These have for the interactions of SP-D with surfactant and with and other glycoconjugates with and organic particulate We for including the of the We also for and for that we aromatic