Differential Ligand Binding Specificities of the Pulmonary Collectins

Jul 18, 2017 - Differential Ligand Binding Specificities of the Pulmonary Collectins Are Determined by the Conformational Freedom of a Surface Loop...
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Differential ligand binding specificities of the pulmonary collectins are determined by the conformational freedom of a surface loop Michael J Rynkiewicz, Huixing Wu, Tanya R. Cafarella, Nikolaos M. Nikolaidis, James Frederick Head, Barbara A. Seaton, and Francis X. McCormack Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01313 • Publication Date (Web): 18 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Differential ligand binding specificities of the pulmonary collectins are determined by the conformational freedom of a surface loop Michael J. Rynkiewicz1, Huixing Wu2, Tanya R. Cafarella1, Nikolaos M. Nikolaidis2, James F. Head1, Barbara A. Seaton1, Francis X. McCormack2* 1

Department of Physiology and Biophysics, Boston University School of Medicine, Boston MA 02118

2

Division of Pulmonary, Critical Care, and Sleep Medicine, University of Cincinnati College of Medicine, Cincinnati OH 45267

*

To whom correspondence should be addressed: Francis X. McCormack, MSB 6165, 231 Albert Sabin Way, Cincinnati 45267-0564. Email [email protected]; Tel. 513-558-4831; FAX 513-558-4858 Funding sources: Carespring Foundation (FXM), Department of Veteran Affairs VA Merit Award (FXM) and NIH/NIAID #PO1-AI083222 (BAS).

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ABBREVIATIONS: dipalmitoylphosphatidylcholine, DPPC; unsaturated phosphatidylcholine, PC; phosphatidylglycerol, PG; phosphatidylinositol, PI; 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 18:0-18:2 PC; lipopolysaccharide, LPS; surfactant protein, SP; carbohydrate recognition domain, CRD; neck and CRD, NCRD; N187S and R197N mutant of SP-A, N; E171D, P175E, N187S and K203D mutant of SP-A, DED; E171D, P175E, N187S, R197N, and K203D mutant of SP-A, DEDN; tensor/libration/screw, TLS; and mannose binding protein, MBP.

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ABSTRACT Lung surfactant proteins (SP) play critical roles in surfactant function and innate immunity. SP-A and SPD, members of the collectin family of C-type lectins, exhibit distinct ligand specificities, effects on surfactant structure, and host defense functions despite extensive structural homology. SP-A binds to dipalmitoylphosphatidylcholine (DPPC), the major surfactant lipid component, but not phosphatidylinositol (PI), whereas SP-D shows the opposite preference. Additionally, SP-A and SP-D recognize widely divergent pathogen-associated molecular patterns. Previous studies suggested that a ligand-induced surface loop conformational change unique to SP-A contributes to lipid binding affinity. In order to test this hypothesis and define the structural features of SP-A and SP-D that determine their ligand binding specificities, a structure-guided approach was used to introduce key features of SP-D into SP-A. A quadruple mutant (E171D/P175E/ R197N/ K203D) which introduced an SP-D-like loop-stabilizing calcium binding site into the carbohydrate recognition domain (CRD) was found to interconvert SP-A ligand binding preferences to an SP-D phenotype, exchanging DPPC for PI specificity, and resulting in loss of lipid A binding, and gain of more avid mannan binding properties. Mutants with constituent single or triple mutations showed alterations in their lipid and sugar binding properties that were intermediate between SP-A and SP-D. Structures of mutant complexes with inositol or methyl-mannose revealed an attenuation of the ligand-induced conformational change relative to wild-type SP-A. These studies suggest that flexibility in a key surface loop supports the distinctive lipid binding functions of SP-A, thus contributing to its multiple functions in surfactant structure and regulation, and host defense.

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Lung surfactant plays a dual physiological role in reducing alveolar surface tension during respiration 1 and serving as the first line of defense against inhaled pathogens2,3. Surfactant is composed of phospholipids, primarily dipalmitoylphosphatidylcholine (DPPC) and lesser components unsaturated phosphatidylcholine (PC), phosphatidylglycerol, and phosphatidylinositol (PI), and proteins including the hydrophobic surfactant proteins (SP) -B and -C, and the hydrophilic surfactant proteins, SP-A and SP-D4– 6 . In the alveolar hypophase, the surfactant components exist as micelle-like aggregates, lattice-like tubular structures and mono- and multilayered membranes7,8. SP-B and SP-C become integrated into surfactant membranes and facilitate their surface spreading and surface-active properties9. SP-A and SPD alternately interact with surfactant membrane interfaces and with conserved motifs on microbial surfaces10. Binding of SP-A to surfactant membranes is important for the structure of tubular myelin7,11, while SP-D regulates surfactant pool sizes and the interconversion between ultrastructural aggregate forms of surfactant12–15. Binding to microbial surfaces results in aggregation and opsonization of organisms to enhance clearance, direct induction of microbial permeability and growth arrest16–18, and modulation of host inflammation2. The structural basis of these divergent functions of the hydrophilic surfactant proteins in surfactant homeostasis is incompletely understood. SP-A and SP-D are structural homologs that belong to the collectin family of C-type lectins. Collectins are defined by a distinctive modular domain structure that includes a disulfide-rich Nterminus, a collagenous region, a coiled-coil neck domain, and a globular carbohydrate recognition domain (CRD) with a calcium ion at the lectin site19,20. Trimers of collectin subunits form by helical folding of their collagen-like domains, and assemble into larger oligomers that comprise a bouquet of tulips-like octadecamer for SP-A and cruciform dodecamer for SP-D21,22. A fragment composed of the neck and CRD (NCRD) of SP-A and SP-D has been widely used as a platform to study the lipid and carbohydrate binding properties of the full-length proteins10,23–26. Ligand specificity appears to be dictated by the CRDs27,28, although many of the finer aspects of collectin-ligand interactions remain incompletely defined. Despite their common domain structure and similar CRDs, SP-A and SP-D have evolved different ligand specificities in line with their particular roles in innate immunity and lipid homeostasis. They have distinctive phospholipid binding specificities: SP-A binds to PC but not PI29,30, while SP-D exhibits the opposite specificity31. The structural basis for the specificity is not readily obvious, since the folding of the the phospholipid binding CRD’s is very similar for the two proteins. However, these alternate ligand preferences almost certainly relate to the differential roles of the pulmonary collectins in tubular myelin formation and surfactant aggregate structure that likely modulate surfactant adsorption to the interface and catabolism, respectively. In the context of host defense, SP-A and SP-D recognize different and often complementary microbial antigens or components, with SP-A ligands generally being more hydrophobic and SP-D ligands richer in carbohydrates. For instance, while both proteins bind to lipopolysaccharides on gram-negative bacteria, SP-A binds preferentially to the lipid A portion whereas SP-D binds to the core saccharides33. Similarly, the binding of SP-A or SP-D to Mycobacterium avium shows interactions with mycobacterial lipid or lipoarabinomannan, respectively34. These different preferences may correlate with distinct host defense functions and serve to extend the range of innate immune surveillance in the lung. We have shown previously that SP-A contains a flexible loop structure in the CRD that is associated with the lectin site and undergoes a large local conformational change with carbohydrate binding35. In these respects, SP-A is dissimilar to other host defense collectins such as SP-D and mannose binding lectin. The same region in SP-D has a more rigid scaffold that shows little or no conformational alteration upon carbohydrate binding24,26,36–39. We proposed that this loop flexibility in SP-A is related to its weaker lectin activity but may increase its affinity for lipids. To test this hypothesis, and to probe the structural origins of their ligand preferences, we engineered selected features of SP-D into SP-A. These 4 ACS Paragon Plus Environment

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included a binding site for new structural calcium ions that would be expected to impart SP-D like rigidity to the SP-A loop24, and a replacement of Arg197 in SP-A by asparagine, which in an analogous position in SP-D facilitates affinity with oligosaccharide ligands. The engineered SP-A proteins show dramatic differences inphospholipid and carbohydrate binding properties, consistent with stepwise conversion of SP-A to a more SP-D-like phenotype. We conclude that SP-A has evolved a long loop structure to specifically bind to PC containing membranes, whereas SP-D has evolved its structure to recognize sugar-containing ligands, including the PI head group, more effectively. EXPERIMENTAL PROCEDURES Reagents-L-α-phosphatidylinositol (Soy PI, 840044C), 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC, 850355C), phosphatidylglycerol (PG 841138), egg phosphatidylcholine (PC), and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0-18:2 PC 850468) were purchased from Avanti Polar Lipids, Inc. Cholesterol, mannan and lipid A were from Sigma. The QuikChange Lightning SiteDirected Mutagenesis Kit was purchased from Agilent Technologies. The pET-30a (+) vector, S-protein horseradish peroxidase (S-protein HRP, 69047), and RosettaBlue competent cells were purchased from EMD4 Bioscience. Fatty acid-free BSA with low immunoglobulin (BAH66-0050) was from Equitech-Bio, Inc. Purified synthetic oligomers were obtained from Eurofins MWG Operon. Expression and isolation of WT and mutant recombinant proteins- A bacterial expression system was used to generate panels of mutant, N-terminally polyhistidine tagged, trimeric fusion peptides comprised of the neck and carbohydrate recognition domain (NCRD) regions of rat SP-A and SP-D proteins for activity assays, following previous published protocols40. Site-directed Figure 1. SP-A NCRD fusion proteins and mutants. Panel A. Schematic mutagenesis was performed diagram of NCRD fusion proteins showing His tag (6X His), S-protein tag, using a QuikChange Lightning enterokinase cleavage site (EK), and the neck and CRD domains with indicated site-directed mutagenesis site of mutations. The N187S mutation removes a consensus N-linked kit. The SP-A NCRD construct glycosylation site. Panel B. SDS-PAGE gel comparing the mobility of the used in previous structural reduced wild-type rat SP-A NCRD (A-NCRD) and rat SP-D NCRD (D-NCRD) studies23,35 and as a template fusion protein with the mobilities of the multi-site or single site substitution for construction of all mutants or used in this study, E171D,P175E,K203D (DED), mutants in these studies has E171D,P175E,K203D,R197N (DEDN) and R197N (arrow). a mutation in consensus glycosylation site that prevents N-linked glycosylation of the CRD (N187S) (when expressed in mammalian expression systems). Additional amino acid changes introduced into the rat SP-A NCRD included the single substitution R197N, or combinatorial mutations E171D, P175E, and K203D (DED mutant) or E171D, P175E, and K203D, R197N mutant (DEDN) (Figure 1). All DNA sequences were verified by automated sequencing of the entire coding sequence of the fusion protein. WT and mutant trimeric neck−CRD domains were expressed in RoseNa Blue competent cells, and were isolated from inclusion bodies. After refolding and oligomerization, the fusion proteins were purified by nickel affinity 5 ACS Paragon Plus Environment

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chromatography, and dialysis in 5 mM Tris-HCl (pH 7.5). Samples were enriched for properly folded proteins by FPLC size fractionation on a Superose 6 column. Protein concentrations were determined using the bicinchoninic acid (BCA) assay with bovine serum albumin as standard. Proteins were tested for calcium dependent binding to carbohydrate linked beads and LPS content was verified to be less than .08 EU/ng protein. For crystallographic studies, to obviate the need for refolding and to facilitate direct comparison to previously solved collectin structures, NCRD mutants of rat SP-A lacking tags and fusion peptides were synthesized in insect cells using baculoviral vectors . Mutant CDNAs were generated using site-directed mutagenesis, as outlined above, and inserted into PVL1392 vectors. Proteins with SP-A or SP-D carbohydrate binding affinities were purified by mannose or maltose Sepharose affinity chromatography respectively, as previously described23. Preparation of Multilamellar PI and DPPC/PG Liposomes- Soy PI or DPPC/PG (w/w 85:15) were dissolved at a concentration of 1 mg/ml in chloroform, dried to a film under nitrogen in a depyrogenated glass tube, and rehydrated for 0.5 h at 37° C in TBS buffer (20 mM Tris-HCl (pH 7.5) and 140mM NaCl). Multilamellar liposomes were generated by vigorous agitation using a vortex mixer, sedimented by centrifugation, and resuspended in TBS buffer before use. Solution phase SP-A/liposome precipitation binding assay- Proteins were preincubated for 15 min at room temperature in TBS buffer with 5 mM calcium chloride or 5 mM EDTA. An equal volume of a DPPC/PG or PI liposome suspension was added, and the mixture was incubated for 60 min at room temperature with shaking. Liposomes and bound proteins were sedimented by centrifugation and the pellets were washed once at room temperature in TBS buffer containing 5 mM calcium chloride or 5 mM EDTA, respectively. Reducing buffer was added to the supernatant and pellet, and after boiling, proteins were resolved by SDS−PAGE and visualized by staining with Coomassie blue. Plates were loaded with an equal volume of supernatant and pellet fractions, incubated overnight, washed, blocked by incubation with TBS buffer containing 1% fatty acid-free BSA, and washed again. An S-protein−HRP conjugate (1:5000 dilution) was added, and bound proteins were detected by addition of peroxidase substrate tetramethylbenzadine (SureBlue Reserve TMB Microwell Peroxidase Substrate [KPL-53-00-02]) and measurement of absorbance at 450 nm. Mannan and lipid A solid phase binding assays. Plates were loaded with mannan (50 µg/ml) or lipid A (10 µg/ml) overnight, blocked for 1 hr at RT with 0.1% (w/v) low-endotoxin, low-immunoglobulin fatty acid-free BSA in TBS, and washed three times. Fusion proteins were added at the indicated final concentration in binding buffer (140 mM NaCl, 20 mM Tris-HCl, 0.1% w/v fatty acid-free BSA) in the presence of 5 mM calcium or 5 mM EDTA and incubated for 60 min at room temperature. After washing, bound fusion proteins were detected using an S-protein−HRP conjugate, as above. Absorbance was measured at 450 nm. Liposome Aggregation- Liposome aggregation experiments were performed as previously described41, with the following modifications. Unilamellar vesicles were produced by probe sonication of lipid mixtures composed of DPPC/ PC /Cholesterol/ 18:0-18:2 PC (ratio to DPPC 1:1: 0.15:0.15) in 10 mM HEPES (pH 7.5) and 150 mM NaCl buffer at 25° C. The liposomes were equilibrated with recombinant mutant Histag SP-As and SP-D (lipid:protein ratio 20:1 by weight) that had been previously crosslinked (to enhance valency for aggregation) with 6X His Tag Antibody Biotin Conjugated Mouse Monoclonal antibody (His tag protein:Histag antibody ratio 2:1 by weight) at 4° C overnight. Aggregation was determined by measuring light scattering (absorbance at 405 nm) at 5 min intervals after the addition of 4 mM calcium (final) and 20 µg/ml of streptavidin. Crystal growth, data collection, and structure solution- The DED and DEDN mutants were crystallized according to protocols previously reported by our group23,35, with the following modifications. Prior to crystallization, the DED mutant (12.5 mg/ml) was incubated with 10 mM calcium chloride and 0.75 mM trimannose at 4°C for 30 minutes. All crystals were grown in hanging drops by 6 ACS Paragon Plus Environment

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mixing equal amounts of protein and reservoir solution on siliconized coverslips and equilibrating over a well of reservoir solution at 17°C. The reservoir solution used for the DED crystals was 10 mM sodium cacodylate (pH 6.0), 80 mM calcium acetate, 0-10% glycerol, and 4-10% (w/v) PEG 20,000. Before freezing for x-ray data collection, DED crystals were soaked in reservoir solution of 10 mM calcium acetate without glycerol, but with stepwise additions of 5, 10, and 15% 2-methyl-2,4-pentanediol with or without (unliganded) either 5% (w/v) inositol or 25% mannose for 10 minutes at each step. The DEDN (17 mg/ml) crystallized from a reservoir solution of 0.2 M calcium chloride and 14% (w/v) PEG 3,350. Before freezing, the DEDN crystals were soaked in reservoir solution supplemented with 1.5 M 1,6hexanediol as a cryoprotectant with or without (unliganded) either 2% (w/v) inositol or 5% (w/v) mannose for 30 minutes before x-ray data collection. All data were collected on a RAXIS-IV++ image plate system with a Rigaku RU-300 rotating anode as the x-ray source. Indexing, integration, and data reduction were performed with the programs DENZO and Scalepack42. To solve the structures, the coordinates of wild type SP-A complexed with mannose (pdb code 35 3PAK ), less the waters and ligands, were used as starting models for Difference Fourier (DED mutant) or molecular replacement (DEDN) to solve the unliganded DED and DEDN complexes. To solve the liganded complexes, the final refined coordinates of the unliganded mutant structures were used as starting models for Difference Fourier. All calculations were carried out using the Phenix package43. In all cases, the starting model structure was completely rebuilt using AutoBuild in Phenix with the starting model excluded from the final structure building steps to reduce phase bias. Iterative cycles of manual rebuilding and refinement were carried out in Coot44 and Phenix until all major peaks in the electron density maps were modeled. In the later stages of the refinements, tensor/libration/screw (TLS) was used to model the temperature factors of the atoms in the model, using 2 zones for refinement corresponding to the neck (the N-terminal residue up to residue 109) and the CRD (residues 110-228). The final structures showed good geometry and agreement to the x-ray diffraction data (Table 1). RESULTS Rationale and design of mutants- Mutations to introduce SP-D features into SP-A were designed using superimposed structures of SP-A and SP-D. First, mutation of R197 to asparagine was predicted to make the lectin sites of SP-A and SP-D identical in their calcium coordination spheres. Second, E171, one of the structural site calcium ligands, is invariant in SP-A. In the superimposition, the end of the E171 side chain occupies the same space as the site 1 calcium in SP-D. Thus, in the DED and DEDN mutants, this side chain needed to be shortened to D, which allows space for the site 1 calcium to bind and provides a favorable residue for coordination to the calcium. In the unliganded SP-A structure, E171 is observed making a salt bridge to the side chain of K203. The position 203 residue in SP-As is either K or Q. Thus, the SP-A side chains of positions 171 and 203 are able to interact with one another, altering the long loop structure, and moving E202 away from the lectin site calcium. Mutation of K203D both removes the potential for interaction with residue 171 and provides a potential site 3 calcium binding site. The fourth mutation used was of P175. The equivalent SP-D residue (E301) participates in both calcium sites 1 and 3. Unlike the previous three residues, P175 does not superimpose well between SP-A and SP-D, since the short loop structures are divergent. However, the mutation of the rigid proline residue to glutamate with the expected binding of calcium was predicted to allow the short loop to adopt a more SP-D like structure. In rat SP-A, residue 175 is proline, but in other SP-As it can be alanine, so the calcium site is the more likely explanation for any observed shift in the short loop structure. Mutant SP-A proteins maintain the collectin fold. To determine whether the DED and DEDN mutants had incorporated the desired structural features from SP-D, the crystal structures were determined. All the

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structures were of good quality and similar resolution to the SP-A structures previously published (Table 1). Table 1 – Data Collection and Refinement Statistics Construct Ligand PDB code Space group Unit cell dimensions (Å, °) Resolution (Å) No. of unique reflections Completeness (%) Mosaicity (°) Redundancy I / σI Data cutoff Rmerge Rwork / Rfree No. of reflections No. of atoms total Protein atoms Calcium and Ligands Waters B factors Protein atoms Calcium and Ligands Waters Bonds lengths (Å) Bond angles (°) Favored (%) Allowed (%) Outliers (%)

DED none 4WR9 P63 a=b=97.190, c=44.629, α=β=90, γ=120 15-2.30 (2.38-2.30) 10829 (1089) 99.9 (99.9)

DED INS 4WUW P63 a=b=97.700, c=45.027, α=β=90, γ=120 15-2.40 (2.49-2.40) 9585 (950) 98.0 (99.9)

DED MAN 4WUX P63 a=b=96.944, c=44.995, α=β=90, γ=120 15-1.90 (1.97-1.90) 18939 (1893) 98.6 (99.8)

DEDN none 4WRC 1 R32 a=b=68.774, c=168.852, α=β=90, γ=120 15-1.80 (1.86-1.80) 14534 (1451) 99.4 (99.9)

DEDN INS 4WRE 1 R32 a=b=66.730, c=165.891, α=β=90, γ=120 15-1.75 (1.81-1.75) 14452 (1304) 98.4 (90.6)

DEDN MAN 4WRF 1 R32 a=b=69.157, c=169.566, α=β=90, γ=120 15-1.9 (1.97-1.90) 12478 (1134) 98.8 (90.1)

1.330 5.6 (4.8) 24.8 (4.0) I ≤ -3σ 0.069 (0.465) 0.1652 (0.2003) 10619

1.243 6.3 (6.3) 20.6 (5.9) I ≤ -3σ 0.083 (0.493) 0.1739 (0.2044) 9569

0.769 5.2 (4.5) 29.9 (3.3) I ≤ -3σ 0.054 (0.507) 0.1723 (0.2022) 18923

0.924 10.7 (9.1) 39.5 (5.6) I ≤ -3σ 0.065 (0.473) 0.2051 (0.2426) 14368

1.386 5.0 (3.4) 21.8 (3.7) I ≤ -3σ 0.083 (0.345) 0.1877 (0.2216) 14272

1.156 5.9 (3.6) 28.1 (3.2) I ≤ -3σ 0.070 (0.397) 0.1981 (0.2440) 12242

1229

1206

1278

1191

1204

1253

1153 3

1137 14

1163 38

1097 11

1094 23

1120 24

73

55

77

83

87

109

49.2 73.4

49.5 71.9

44.8 59.5

32.3 34.6

22.3 27.3

37.1 38.3

47.7 0.013

48.6 0.018

48.6 0.020

34.6 0.011

30.4 0.016

45.0 0.016

0.759

1.021

1.539

1.208

1.022

0.936

97.22 1.39 1.39

96.48 2.82 0.70

96.55 2.76 0.69

95.65 3.62 0.72

97.06 2.94 0.00

95.74 3.55 0.71

1

The hexagonal setting of the R32 space group was used.

Interestingly, while the DED mutant crystallized in the same space group as wild-type SP-A, the DEDN mutant crystallized in a new space group; however, the trimeric structure of the mutants was maintained. As with the wild type SP-A crystal structure, the asymmetric unit of the DEDN mutant consists of a monomer, but the trimer was recreated using crystallographic symmetries, despite the different space groups. In the mutations, the overall fold of the protein is conserved (see Supplementary 8 ACS Paragon Plus Environment

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Data). For example, the root-mean-square-deviation (r.m.s.d.) of fit for the mannose-bound mutant structures to the corresponding published SP-A structure35 ranged between 0.70 Å and 0.76 Å (r.m.s.d. calculated using NCRD alpha carbons). The R197N mutant failed to crystallize under any conditions, perhaps due to destabilization of the SP-A lectin site in the absence of the DED mutations. Mutant proteins incorporate structural calcium sites- Analysis of the DED and DEDN unliganded and liganded structures revealed three calcium sites (sites 1-345) in the NCRD: one lectin site calcium (site 2) and two other calciums (sites 1 and 3) between the long (residues 181-204) and short loops (residues 171-177). In the mutant crystal structures, there is a site 1 calcium located near the E171D mutation site (see Supplemental Data), where it is coordinated by five residues. The side chain oxygens of D171 as well as the backbone oxygen of E202 are coordinating calcium in all mutant complexes (see Supplemental Data), and E175 (the P175E mutation site) is coordinating in almost all complexes. Other residues making interactions in various complexes include the side chains of Q199, and D215 as well as the backbone oxygen of Q199. Also observed in the structures is a site 3 calcium located near the K203D mutation site (see Supplemental Data), where the calcium is coordinated by the side chain of D203 and the backbone oxygen of G200 in the unliganded DED and DEDN/mannose structures (see Supplemental Data). While the B factors and occupancies of sites 1 and 3 are poor, the environment around the sites precludes modeling the observed electron density peaks as water. Interestingly, the short loop conformations in the DED and DEDN structures are closer to the SP-D backbone (r.m.s.d. = 0.6-1.8 Å, calculated using backbone atoms of residues 170-178) than wild-type SP-A (r.m.s.d. = 2.3-2.8 Å), showing that the structures of the long and short loops have incorporated features that are like SP-D instead of SP-A. Since the mutated residues appear to play a key role in coordination of the novel calcium sites, the incorporation of these novel calcium sites into the SP-A proteins that are not present in the wild type protein appear to be a consequence of the mutations. Therefore, the crystal structures show that the DED and DEDN mutants contain the structural calcium sites 1 and 3 observed in SP-D, however, the sites are not identical. For instance, the interaction of the backbone carbonyl of G200 in the SP-A mutants is not observed in SP-D, possibly due to the shorter long loop in SP-D not being able to reach site 3. Mutant SP-A proteins show altered lectin site calcium coordination- The site 2 calcium is located between the long loop and one of the beta strands in the NCRD (Figure 2). Fig. 2. Ligand binding region of SP-A, SP-D, and mutants. Ribbon diagram of Both DED and the long and short loops of wild type SP-A, SP-D, and the mutant constructs, DEDN have mostly showing lectin site and structural calcium sites. Comparisons are made identical features of the between unliganded and the corresponding mannose complexes (liganded). calcium coordination. The Mannose molecule from complex is shown over the lectin calcium. Calcium side chains of E195, E202, sites are numbered as in SP-D. N214, and D215 as well as the backbone oxygen of D215 coordinate the calcium in both mutants. In addition, two other coordinating interactions are provided by either water molecules (unliganded) or oxygens from the sugar ligand (mannose and inositol complexes). A key difference between the mutants is residue 197. In DED, this residue is arginine, as in wild type SP-A, and the backbone carbonyl is observed in the structures making coordination interactions to the site 2 calcium. In DEDN, residue 197 has been mutated to asparagine, as in SP-D, and in crystal structures of DEDN the side chain of N197 is 9 ACS Paragon Plus Environment

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coordinated to the site 2 calcium. Notably, the R197N mutation alters the lectin site so that it is identical to the lectin site in SP-D (r.m.s.d. of fit = 0.2-0.4 Å calculated using the lectin site calcium and all non-hydrogen atoms of the coordinating protein residues for the unliganded, inositol, and mannose complexes). Mutant-ligand complexes- Structures were solved of DED and DEDN in complex with one of two saccharide ligands, D-mannose or D-myo-inositol. Both ligands are bound via equatorial vicinal diols in the C-lectin sites of the mutants, where two of the ligand hydroxyl groups displace two waters observed in the unliganded structures. In the case of inositol, the sugar is making coordination interactions to the calcium through its O1 and O6 hydroxyls, whereas in the mannose complexes the sugar is bound through O3 and O4. For both mutants, additional hydrogen bonding interactions to the lectin site-bound hydroxyls of the ligands are made by the side chains of E195, E202, and N214, similar to other collectin structures. For DEDN, an additional hydrogen bond is made by the side chain of N197, an interaction that is unique to DEDN since the arginine of the wild type and DED constructs cannot make this bond. Neither ligand makes any other direct interactions to the protein outside of the lectin site. Soaking of inositol into wild type SP-A crystals under similar conditions to the ones used here showed no significant occupancy of the lectin site. While only one inositol molecule was found in the mutant complexes, there are three mannoses in the DED/mannose complex. The two outside the lectin site are bound in areas of the molecule not previously implicated in sugar binding. The first site is located near to the disulfide bond that defines the ends of the long and short loops. The mannose makes hydrogen bonds to the side chain of D129, as well as the mutated side chain of D203. The second site is located on the underside of the CRD, in a pocket formed by N141, I142, V144, R146, G168, Y180, L181, D182, and E227, where it makes several hydrogen bonds. It is not clear if there is any biological significance to these sites, since they do not appear in the wild type or DEDN structures and the concentration of mannose used in the experiments is very high. DED and DEDN mutations alter calcium dependent phospholipid binding preference of SPA from DPPC to PI- As Figure 3. Phospholipid binding assay. Panel A. Liposome binding assay. expected, phospholipid Multilamellar liposomes composed of DPPC/PG or PI were mixed with NCRD precipitation binding data fusion proteins in the presence of calcium or EDTA as indicated. The proportion shows that SP-A binds to of protein bound to phospholipid was determined by centrifugation and visual DPPC liposomes but not inspection of collectin species in the supernatant (sn) and pellet fractions after PI liposomes, while SP-D resolu\on by SDS−PAGE. Panel B. Binding assay was conducted as above, but in has the opposite the absence of liposomes. Panel C. Supernatant and pellet fractions from specificity, binding to PI experiments in Panel A were also coated onto 96 well plates overnight, but not to DPPC (Figure incubated with blocking buffer and washed. Bound fusion proteins were 3). Introduction of DED detected by addition of S-protein−HRP conjugate and TMB substrate. substitutions into SP-A Absorbance was measured at 450 nm. Data are mean ± S.E., n=3, *p R197N his-tagged N-CRD fusion proteins and anti-His tag biotinylated 15.4% ± 1.1. These findings antibody. Aggregation was determined by measuring light scattering clearly indicate that stepwise +2 (A=405 nm) at 5 min intervals after the addition of 4 mM Ca (final) mutations result in incremental and 20 µg/ml of streptavidin. Panel B. Experiments were conducted as impairment of DPPC liposome in A. but without liposomes. aggregation by DEDN. DEDN mutations reduce lipid A binding while DED and R197N mutations have no effect- SP-A has a binding preference for lipid A, a ligand that SP-D does not recognize46. To test whether the SP-A mutant constructs have altered lipid A binding, solid phase binding of R197N, DED and DEDN to lipid A was measured (Figure 5). The data showed that the DED and R197N mutants bind to lipid A with an affinity that is similar to wild type SP-A. The binding is calcium independent; an SP-A/LPS interaction characteristic that has been reported by some groups32 but not others47. However, DEDN mutations to SP-A diminish lipid A binding affinity, with total binding reduced by nearly 2-fold relative to wild-type SP-A NCRD. This is the expected result given the more SP-D-like nature of the DEDN construct. DEDN and N (R197N) mutations to SP-A enhance lectin binding. In light of the changes in lipid binding Figure 5. Solid Phase Lipid A Assay. Lipid A coated plates were loaded with preferences, the lectin binding NCRD fusion proteins in binding buffer containing 5 mM calcium (Panel A) abilities of the SP-A constructs or 5 mM EDTA (Panel B). After washing, bound proteins were detected were also examined. SP-A is a using S-protein-linked-HRP and TMB substrate as described in Materials and relatively poor lectin and in our Methods. Data are mean ± S.E., n=4. 11 ACS Paragon Plus Environment

Biochemistry

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hands did not bind to mannan in the solid phase assay, whereas SP-D is a strong lectin and bound well to mannan. Therefore, mannan solid phase direct binding experiments were performed to measure the mannan affinity of wild type SP-A NCRD, wild type SP-D NCRD and the three SP-A NCRD Figure 6. Mannan solid phase binding assay- Mannan coated plates were mutants, in an effort to loaded with NCRD fusion proteins in binding buffer containing 5 mM determine the effect of the calcium (Panel A) or 5 mM EDTA (Panel B). After washing, bound fusion mutations on lectin activity proteins were detected by addition of S-protein-linked-HRP as described in (Figure 6). The R197N Materials and Methods. Data are mean ± S.E., n=4. mutation confers avid binding to mannan, exceeding even that of SP-D. The DEDN mutant exhibits saturable, calcium-dependent binding to mannan, similar to wild type SP-D NCRD. In contrast, the DED mutant and SP-A NCRD do not well bind to mannan. We conclude the lectin binding ability is solely affected by mutations at the lectin site, namely the R197N substitution, but the preference for binding to the phosphatidylglyceride, PI, requires stiffening of the long loop. DISCUSSION Prior studies have shown that lipid binding and ligand preferences of surfactant proteins A and D are contained primarily within their CRDs. Studies of chimeras implicated the CRD rather than other protein domains in the binding preference of SP-A for DPPC over PI, which is preferred by SP-D28. Subsequently, studies employing chimeras of the NCRDs27 demonstrated that replacement of residues 195-228 could interconvert ligand phospholipid binding preferences; however, chimeras in which almost 2/3 of the NCRD was replaced were required to fully switch the specificity of SP-A from DPPC to PI. The importance of R197 in lipid binding has been shown by mutational analysis48. In these studies, the R197D and R197N mutants showed alterations to the ability of SP-A to aggregate DPPC liposomes. In other studies, an SP-A double mutation (E195Q/R197D) that changed the mannose specificity to galactose produced a mutant that would bind but not aggregate DPPC liposomes41. Further, alanine substitutions of several lectin site residues showed a loss of DPPC liposome binding and aggregation49. Overall, these data implied a critical role for the CRD lectin site in determination of lipid binding preferences of SP-A. However, since no structural data were available at the time of these early studies, no conclusions could be drawn about the structural determinants of lipid specificity. Surfactant proteins A and D are close structural homologs, and they both bind saccharide and phospholipid ligands, yet they are mechanistically distinct in important ways. Several lines of evidence suggest that SP-D has evolved a structure that prefers carbohydrates, such as the saccharide moieties of complex ligands such as lipopolysaccharide (LPS) and phosphatidylinositol (PI)31,46, whereas SP-A binds lipid ligands more effectively. The structural basis of the interactions between collectins and their ligands is best understood for SP-D. Crystal structures of complexes between SP-D and myo-inositol and inositol-1-phosphate show that the inositol ring binds to the lectin calcium in the usual C-lectin mode, i.e. with two vicinal equatorial ring oxygens coordinating the calcium ion39. Similarly, the SP-D lectin calcium also binds to heptoses from the conserved core of LPS38. SP-A, on the other hand, binds 12 ACS Paragon Plus Environment

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Biochemistry

preferentially to lipid A and PC, neither of which contains a saccharide with the necessary vicinal equatorial oxygens for binding the lectin calcium. A further difference between the two surfactant proteins is that SP-D undergoes no significant structural alteration upon binding saccharides24,26,36–39, whereas SP-A undergoes a significant ligand-induced conformational change involving the C-lectin site as well as the long loop residues near the lectin site35. Taken together, these observations imply that the lectin site of SP-A is mechanistically distinct from that of SP-D when it comes to lipid binding. Observation of two distinct structural conformations in SP-A lead to the proposal that the liganded conformation binds sugars as SP-D or MBP do, allowing SP-A to perform basic lectin functions, but that the unliganded form was better able to bind to DPPC membranes, allowing SP-A to perform functions such as supporting the formation of tubular myelin. Similar functional distinctions have been drawn for other lectins that undergo large conformational changes. For human CD23, a low affinity IgE receptor, it was proposed that the observed conformational change would allow CD23 to bind at nonglycosylated sites on the protein50. For P-selectin, a platelet cell adhesion protein, the observed conformational change was proposed to act as a switch between a low affinity and high affinity form, which was thought to be important in the initial tethering and rolling of leukocytes by P-selectin51. Thus, there is some precedent for conformational changes in C-type lectins resulting in expanded functionality for the proteins. In order to validate this hypothesis for SP-A, we set out to alter the protein in such a way that the long loop would be made less flexible and locked in the ligand-bound conformation; our hypothesis was that if correct, this alteration would lead to lessening of DPPC binding and an increase in sugar binding. In our engineering design, we incorporated into SP-A structural features from SP-D that were related to its overall structural rigidity around the lectin site. We focused on residues involved in calcium binding. First, we mutated residues in the SP-A lectin site to produce the R197N mutant, where the Asn corresponds to Asn323 of the conserved EPN motif of SP-D. The Asn side chain is critical in the SP-D lectin site, since it makes coordination interactions to the lectin site calcium, but also interacts with the sugar ligand. Second, we focused on the structural calciums present in collectins other than SP-A. These calciums are located between the long and short loops in SP-D and appear to stiffen its long loop structure. Therefore, key calcium binding residues were incorporated into the SP-A structure to produce the E171D, P175E, and K203D triple mutant (termed the DED mutant). Lastly, the DEDN mutant was constructed that would have both lectin site (R197N) and structural calcium site mutations (DED) incorporated. We performed crystallographic analysis of the DED and DEDN mutants and compared these with wild-type SP-A and SP-D. The crystal structures of the SP-A mutants show successful incorporation of novel calcium sites and greater structural similarity to SP-D (see Results for details). We then compared these mutants in complex with mannose or inositol. Binding of these ligands was structurally similar in these complexes except that wild-type SP-A did not bind inositol and the DED mutant bound it poorly, as evidenced by weak electron density. However, DEDN bound inositol well, similar to SP-D. Importantly, the crystallographic analysis revealed that the ligand-induced SP-A conformational change was attenuated in the DED and DEDN mutants (Figure 2). The root-mean-square-deviation (r.m.s.d.) comparing backbone atoms in the unliganded and mannose-complexed wild type SP-A is 3.3 Å in the loop most closely involved in the transition (residues 197-203), whereas the same r.m.s.d. in SP-D is 0.32 Å. The corresponding values for the DED (1.6 Å for inositol and 2.0 Å for mannose) and DEDN (1.4 Å for both inositol and mannose) mutants are considerably lower than that of wild-type SP-A, consistent with the lack of a significant conformational change in the presence of ligand. The incorporation of the structural calciums by mutation into SP-A has the effect of attenuating some of the flexibility of the long loop observed in wild-type SP-A structures. Comparison between DED and DEDN suggests that the attenuation is greater when both the lectin and structural calcium sites are altered.

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Biochemistry

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In addition to the structural analysis, we investigated sugar (mannose) and lipid (PI, DPPC, or lipid A) binding properties of the DED, DEDN, and R197N. We found dramatic changes, particularly with lipid ligands, that corresponded to a switch from the SP-A to SP-D phenotype. A major finding was that, in sharp contrast to the SP-A wild-type, the DEDN mutant bound exclusively to PI and not DPPC. The DED and R197N mutants bound to both PI and DPPC reflecting intermediate properties between the SP-A and SP-D phenotypes. They also showed reduced ability to aggregate liposomes despite avid lipid binding; a profile that had been reported for the E195Q, R197D SP-A variant, perhaps related to effects of mutations on orientation of the CRDs on the liposomal surface that affect lateral protein-protein interactions between CRDs41. All of the proteins that bound DPPC, i.e. wild-type SP-A and the DED and R197N mutants, were capable of binding lipid A in a calcium independent manner. It should noted although not required for binding, calcium has been reported to promote the formation of SP-A/LPS aggregates, and in the presence of SP-A to influence the distribution and behavior of lipids in mixed DPPC/LPS membranes32,52. As expected, binding of lipid A by the SP-D-like DEDN was significantly decreased. Taken together, these studies correlate the structural conformational change in SP-A with the ability to bind to DPPC. In contrast to lipid binding, the binding of sugars by the mutants seemed to be affected only by the lectin site alterations. SP-A and the DED mutant bound mannan poorly. However, mutation of R197N was sufficient to confer mannan binding affinity in the solid phase assays that was similar to SPD. Unlike the lipid binding studies, mannan binding was not further increased by addition of the DED mutations to the R197N construct, i.e. DEDN binds mannan similarly to R197N and SP-D. With respect to lectin activities, the flexibility of the long loop appeared to be less critical than the coordination of the lectin site calcium. However, the importance of the long loop in sugar binding has been suggested in studies of other collectins. Attempts to convert the specificity of mannose binding protein (MBP) from mannose to galactose required more than just the simple replacement of the consensus EPN sequence with QPD53. To fully convert the specificity, a point mutation and the insertion of amino acids from the asialoglycoprotein receptor into the long loop of MBP were required54. Another example of loop involvement in ligand binding is porcine SP-D. Compared with human SP-D, pig SP-D contains an additional three residues in its long loop. Crystallographic and molecular dynamics analysis of the pig SPD structure showed that these three residues are able to contact branching mannoses on oligomannose ligands. This ability was proposed as a mechanism to explain the greater affinity of pig SP-D for some oligosaccharides compared to human SP-D25. From the present data, conclusions can be drawn as to the functional consequences of the SP-A mutations. The incorporation of SP-D-like structures and selectivities into SP-A in these studies was performed by mutation of only four residues - R197, E171, K203, and P175. The structural calcium sites associated with the DED mutation appear to be critical in defining the lipid selectivity of SP-A, since R197N is not by itself sufficient to switch over the membrane binding preference to PI or to abolish lipid A binding. In contrast, the R197N mutation enhances the lectin functions of the protein, as observed here by the low mannan binding affinity of SP-A and the DED mutant compared with SP-D and DEDN. The crystallographic data suggest a structural basis for the effects of this substitution. Position 197 in SPA from various species is either arginine or alanine, in contrast to other collectins which have asparagine. Of these, only asparagine can make lectin site calcium interactions as observed in SP-D or the DEDN mutant. We speculate that the evolutionary advantage of the conserved Glu-Pro-Ala/Arg motif that differentiates SP-A from all other collectins (which have Glu-Pro-Asn in that position) is preferential binding to lipid over carbohydrate, leading to front line-positioning of SP-A on membranes lining the air-liquid interface. It is also possible that these sequence differences confer differential binding of SP-A and SP-D to a cellular receptor, or other function that was not tested here. In summary, the present studies permit a parsing out of the precise structural features that alter lipid specificity in SP-A. The data show that the inherent conformational flexibility of the SP-A lectin site 14 ACS Paragon Plus Environment

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Biochemistry

region is hindered in SP-D by the rigidity imposed by its structural calciums. The DED mutation recreates this rigidity and introduces PI binding into SP-A. The Arg197 mutation improves the ability of SP-A to bind sugars and switches lipid specificity from DPPC to PI. In all of these respects, the four-residue mutation used in this study produces an SP-D-like phenotype in SP-A. The combined data also support the hypothesis that the conformational flexibility of SP-A structure in the lectin region facilitates DPPC binding while curbing PI binding. These results are consistent with our hypothesis that SP-A flexibility is required for optimal interactions with a dynamic ligand such as membrane lipids and other lipid aggregates such as tubular myelin. On an evolutionary level, relatively few mutations in a common ancestral collectin may have extended the range of microbial targets of this class of proteins, enhancing host adaptation and survival.

Supportive information- Diagrams of collectin structures used in this study, including views of all CRD calcium coordination sites and lectin domain calcium coordination sites.

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For table of contents use only

Differential ligand binding specificities of the pulmonary collectins are determined by the conformational freedom of a surface loop Michael J. Rynkiewicz1, Huixing Wu2, Tanya R. Cafarella1, Nikolaos M. Nikolaidis2, James F. Head1, Barbara A. Seaton1, Francis X. McCormack2*

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