Structural Characterization of a Mannose-binding Protein–Trimannoside Complex using Residual Dipolar Couplings

doi:10.1016/S0022-2836(03)00268-7

J. Mol. Biol. (2003) 328, 451–462

Structural Characterization of a Mannose-binding Protein –Trimannoside Complex using Residual Dipolar Couplings Nitin U. Jain1, Schroeder Noble2 and James H. Prestegard3* 1

Department of Biochemistry and Cellular and Molecular Biology University of Tennessee Knoxville, TN 37996-0840 USA 2

Department of Chemistry University of North Carolina Chapel Hill, NC 27599-3290 USA 3 Complex Carbohydrate Research Center University of Georgia 220 Riverbend Road, Athens GA 30602-4712, USA

*Corresponding author

The ligand-binding properties of a 53 kDa homomultimeric trimer from mannose-binding protein (MBP) have been investigated using residual dipolar couplings (RDCs) that are easily measured from NMR spectra of the ligand and isotopically labeled protein. Using a limited set of 1H – 15N backbone amide NMR assignments for MBP and orientational information derived from the RDC measurements in aligned media, an order tensor for MBP has been determined that is consistent with symmetrybased predictions of an axially symmetric system. 13C – 1H couplings for a bound trisaccharide ligand, methyl 3,6-di-O-(a-D -mannopyranosyl)-a-D mannopyranoside (trimannoside) have been determined at natural abundance and used as orientational constraints. The bound ligand geometry and orientational constraints allowed docking of the trimannoside ligand in the binding site of MBP to produce a structural model for MBP – oligosaccharide interactions. q 2003 Elsevier Science Ltd. All rights reserved

Keywords: NMR; residual dipolar couplings; mannose-binding protein; trimannoside; protein– carbohydrate interactions

Introduction The determination of the bound geometry of ligands in protein-binding sites is often prerequisite to rational design of molecules that can mimic or inhibit the action of these ligands. Crystal structures often provide data for specific ligands, but it is useful to have methods that can readily handle a variety of ligands and do this in a native solution environment. NMR provides one such method, most frequently through the generation of nuclear Overhauser effect (NOE) data.1,2 Transferred NOEs between protons on the ligand give bound geometries effectively, but placement of the ligand on the protein surface using NOE data requires extensive assignment of protein resonances and measurement of NOEs between ligand and protein Abbreviations used: MBP, maltose-binding protein; FID, free induction decay; NOE, nuclear Overhauser effect; TROSY, transverse relaxation optimized spectroscopy; CE-TROSY, coupling-enhanced TROSY; RDC, residual dipolar couplings; HSQC, heteronuclear single quantum coherence; CRD, carbohydrate recognition domain; AMM, a-methyl mannose. E-mail address of the corresponding author: [email protected]

protons. However, assignment of resonances and measurement of the intermolecular NOEs is often difficult for larger proteins. Moreover, measurement of NOEs is often prohibited when there are few short-range contacts between non-exchangable protons, as in carbohydrate-binding proteins, where hydrogen bonding interactions dominate ligand recognition. Here, we illustrate an alternate approach dependent on the use of residual dipolar couplings (RDCs) from 13C – 1H pairs on a ligand to deduce a preferred orientation for the ligand, and the use of protein symmetry properties to deduce the corresponding orientation for the protein. The protein is mannose-binding protein (MBP), a lectin with well defined crystal structure, and the ligand is methyl 3,6-di-O-(a-D -mannopyranosyl)-a-D -mannopyranoside (trimannoside), a trisaccharide common to N-glycosides. The use of RDCs in NMR structure determination protocols has been well documented.3 – 5 RDCs provide long-range structural information in the form of orientational constraints that is complementary to that of short-range NOE distance constraints. RDCs measured in partially aligned media have been applied primarily to structure refinement of proteins and determination of domain orientation in proteins.6 – 8 In the latter

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

452

Figure 1. X-ray crystal structure of the MBP trimer consisting of the carbohydrate recognition domain and coiled coil helical region.22 The 3-fold rotational axis of symmetry is seen passing through the center of the molecule. Calcium atoms are shown in red and the chlorine atom is shown in green. The Figure was prepared using the program MolMol.33

studies, sufficient dipolar couplings between magnetic nuclei at fixed distances (such as a 15N – 1H pair) are measured within a structurally well defined domain to determine the principal order frame as seen from the point of view of a domain. When domains combine to form a single rigid structure, they must share a principal order frame. Hence, alignment of order frames determined for each domain provides a constraint on structural assembly without the necessity of close approach of protons at the domain interfaces. Similar constraints would arise in the assembly of a ligand –protein complex if sufficient data could be collected on both the protein and the ligand. However, application of RDCs to protein – ligand interactions have been few,9 – 11 partly due to the necessity of separately determining the orientation of large proteins, and partly due to the difficulties in producing isotopically labeled forms of complex ligands. In the case of large proteins, difficulties arise because interpretation of RDCs requires structural information and assignment of the protein in question. While this task is less than that for NOE-based methods, because assignments can be confined to backbone resonances, the task can still be formidable. Utilization of the inherent symmetry properties of a homomultimeric protein system represents one way of reducing the need for assignments and RDC data on the protein. Bolon et al. have described the application of a symmetry-aided approach in a study involving binding of a small ligand, a-methyl mannose (AMM), to MBP.9 MBP is found mainly in two forms, MBP-A in the

Mannose-binding Protein –Trimannoside Complex

serum and MBP-C in the liver. Both belong to a group of mammalian lectins known as collectins that is involved in the innate immune response of mammals.12,13 MBP mediates the recognition of specific high-mannose oligosaccharides on the cell surfaces of invading pathogenic bacteria and yeasts. The binding of MBP to these cell surfaces then activates a complement cascade leading to the destruction of pathogens and providing a defense mechanism for the host.14 It has been suggested that dysfunction of MBP in this process plays a role in certain inflammatory diseases.15 Early studies on MBP indicated that MBP-C primarily recognizes the core structure of complex oligosaccharides, whereas MBP-A recognizes only the terminal sugar of these structures.16 Also, MBP-C has a higher affinity for oligosaccharides than MBP-A. It is likely that these specific differences in binding may be functionally important, and this has stimulated a number of investigations into specific-binding properties.17 – 21 Structurally, MBP is a homotrimer that exhibits a 3-fold axis of symmetry passing through the center of an a-helical coiled-coil region holding three carbohydrate-recognition domains (CRDs) in place (Figure 1).22 For systems with a 3-fold or higher symmetry axis, it has been shown that the order tensor must be axially symmetric and have its principal axis coincident with the 3-fold axis.23 Thus, with the availability of a structure displaying appropriate symmetry properties, it seemed possible to entirely avoid NMR data collection on the protein for purposes of protein– ligand studies. The previous NMR study of MBP-A demonstrated this principle, but also found inconsistencies between NMR-derived and crystal-derived ligandbinding geometries, dictating the need for further exploration.9 To be more specific, the previous study of AMM bound to the MBP-A trimer showed that the ligand displayed behavior characteristic of participation in a complex with a 3-fold rotational symmetry axis, but the binding geometry of AMM as determined using RDC measurements differed from the geometry of bound AMM in the X-ray structure of the closely homologous MBP-C through a rotation of about 408.9 Several plausible explanations for this observed difference were offered. It is possible that the binding geometry for AMM in MBP-A is different from that in MBP-C. It is possible that the actual geometry of the MBP-A trimer in solution is different from that depicted in the crystal structure due to displacement of CRDs relative to the 3-fold symmetry axis. And, it is possible that mobility in the binding site, or averaging between binding sites, leads to the apparent variations. The latter possibility is unlikely, since it is difficult to introduce large internal motions that will not destroy the order characteristic of a 3-fold symmetric system. On the other hand, the possibility of deviations between crystal structures and solution structures is supported by deviations seen among various

Mannose-binding Protein –Trimannoside Complex

453

Figure 2. (a) The 15N– 1H TROSY spectrum of a 0.7 mM sample of 15N,2H (50% randomly deuterated)-labeled MBP trimer and (b) 15N – 1H HSQC spectrum of a 1 mM sample of 15N-labeled MBP dimer. Both spectra were recorded on a Varian Unity INOVA 800 MHz spectrometer at 25 8C and under identical sample conditions (25 mM Tris – HCl (pH 7.8), 50 mM NaCl, 10% 2H2O). Note that resolution is very similar despite the factor of 2 difference in molecular mass. Assignments for some selected residues in the trimer for which RDC measurements were obtained are shown in (a). Residues 155, 166 and 180 are not shown, since the peaks for these residues lie outside the spectral region shown.

MBP trimeric constructs in crystals,19,24 and this possibility is worth investigation. We have therefore investigated the symmetry properties and orientation of an order tensor fixed in the coordinate frame of a single CRD of the MBP-A form from rat (referred to here as MBP) by assigning resonances from a 15N labeled MBP sample, measuring RDCs for a subset of these resonances, and using them to calculate the order tensor. This same order tensor was subsequently used to obtain relative orientation of a new trisaccharide ligand, methyl 3,6-di-O-(a-D -mannopyranosyl)-a-D -mannopyranoside (trimannoside) (Figure 7(a)) bound to MBP and dock it onto the binding site of MBP. The use of this new ligand gives us the opportunity to compare solution results with recently published crystal structures of MBP that have a number of different ligands bound.21 It also gives us the opportunity to demonstrate that it is possible to collect RDC data for a ligand without the need for 13C enrichment. This will provide a further test of the validity of the methods and demonstrate potential for applicability to a much larger range of ligands.

Results and Discussion Measurement of RDCs and backbone assignments for MBP The high molecular mass of MBP makes it difficult to obtain high-resolution solution spectra due to considerable line-broadening. It is well known that significant line narrowing can be obtained by employing deuteration in concert with the trans-

verse relaxation optimized spectroscopy (TROSY) experiment.25 As is often the case with other proteins, MBP yields from expression in Escherichia coli in highly deuterated media are low. A possible compromise between sufficient protein yield (and also expense) and narrower line-widths can be obtained by using moderate levels of deuteration. A 15N-labeled sample of MBP randomly deuterated to an extent of 50% was therefore prepared and used for performing RDC measurements. Use of such a sample gave good quality TROSY spectra, as seen in Figure 2(a). Although the partially deuterated sample was suitable for RDC measurements, the increased relaxation effects inherent in a large protein system were still too significant to obtain an adequate number of sequential connectivities for backbone triple resonance assignments from three-dimensional experiments. We therefore decided to make use of a dimeric form of MBP that retains the structural features of the trimer as evidenced by the minimal chemical shift changes for the majority of residues in the dimer versus the trimer (Figure 2(b) compared to (a)). Such a dimer has been crystallized by Weis and co-workers,26 and consists only of the CRD without the coiled-coil neck region. The CRD structures are similar enough in the dimer and trimer for the dimer assignments of the CRD to be transferred to the trimer with a high degree of certainty. The use of the dimer for assignment purposes reduced the effective molecular mass of the protein to almost one-half, greatly improving the line-widths and sensitivity. This allowed the rapid assignment of approximately 50% of the backbone 1H, 15N, 13Ca and 13Cb resonances. These assignments were then transferred

454

Mannose-binding Protein –Trimannoside Complex

Figure 3. The 2D CE-TROSY pulse sequence for measurement of 15 N – 1H RDCs in large proteins. The sequence incorporates an extra coupling evolution period controlled by the parameter k (varies between 0 and 1). The 15N chemical shift evolution occurs during the period t1 : Open bars represent 908x pulses, while filled bars represent 1808x pulses unless noted otherwise. The final proton 1808 pulse is a composite pulse consisting of the sequence 908x(selective) 2 18082x(nonselective) 2 908x(selective). t was set to a value of 5.2 ms. Sensitivity enhancement is achieved using a two-part phase cycle with the first FID recorded with f1 ¼ ðþy; 2y; 2x; þxÞ; f2 ¼ þy; f3 ¼ þx; f4 ¼ ðþx; 2x; þy; 2yÞ and the second FID recorded with f1 ¼ ðþy; 2y; 2x; þxÞ; f2 ¼ 2y; f3 ¼ 2x; f4 ¼ ð2x; þx; þy; 2yÞ: The data were processed using the sensitivity-enhanced scheme of Rance.31 The gradients used are: G1 ¼ (500 ms, 5 G/cm); G2 ¼ (300 ms, 4 G/cm); G3 ¼ (500 ms, 5 G/cm); G4 ¼ (500 ms, 12 G/cm).

to the trimer and utilized along with RDC measurements for determination of the order tensor for the trimer. Yet another difficulty was encountered during the process of RDC measurements for a protein of the size of MBP. Normally for proteins, 1H – 15N RDCs are measured as differences in splittings of doublet components in the 15N dimension of 1 H – 15N J-coupled spectra acquired in aligned and isotropic media, or as the difference in positions of

doublet components seen in pairs of spectra acquired using the IPAP scheme.27 However, 1 H – 15N J-coupled spectra or IPAP spectra for proteins . 30 kDa suffer from severe line-broadening for the upfield component due to cross-correlated relaxation effects. This makes it very difficult to measure the line position of the upfield component accurately, leading to significant errors in measurement. To retain some of the advantages in linewidth of the narrower, TROSY peak, and to gain

Figure 4. RDC measurements from spectra recorded using the CE-TROSY sequence in Figure 3 at 800 MHz on a 0.7 mM sample of 15N,2H (50% randomly deuterated)-labeled sample of the MBP trimer. A 6% (w/v) bicelle solution was used for collection of data in the aligned phase. J-coupling values are measured from the difference in peak positions in the pair of spectra for the isotropic and aligned phase, and are scaled depending on the value of k. The scaled values are shown within parentheses for two representative peaks (Thr160 and Asn205).

Mannose-binding Protein –Trimannoside Complex

Figure 5. Sauson –Flaumsteed projection of the directions of ordering (Szz, Syy and Sxx) for oriented MBP. The direction of highest order Szz is coincident with the rotational symmetry axis as viewed with respect to the molecular frame of trimeric MBP (shown as x; y; z in the Figure).

an ability to manipulate positions to reduce accidental overlap, we introduce a new experimental scheme utilizing the TROSY principle to carry out RDC measurements. The TROSY experiment, which we denote as coupling-enhanced TROSY (CE-TROSY), is shown in Figure 3. It utilizes the well-known elements of accordion spectroscopy used previously to measure 1H – 15N J-couplings in a 1H – 15N CE- heteronuclear single quantum coherence (HSQC) experiment.28 A similar TROSYbased scheme for measurement of RDCs, incorporated as part of a 3D HNCO experiment, has been used by Kay and co-workers.29 Our sequence differs slightly from their sequence in that it is a simple 2D experiment that includes a water flipback element.30 The evolution periods are optimized for minimal relaxation and prevention of radiation damping by placing a pair of water suppression gradients within the t1 period.31 Although additional line-broadening in the CETROSY peaks is observed as a result of mixing of TROSY and anti-TROSY peaks due to inclusion of the extra coupling evolution period in the sequence, the observed line-broadening could be

455

held to a reasonable level by carefully choosing an appropriate value of k (usually between 0 and 1), as has been suggested by Kay and co-workers.29 Example data from isotropic and aligned media are shown in Figure 4 for amide resonances from a pair of amino acid residues that happen to fall in the same spectral region. Between the limited assignment of backbone resonances and ability to resolve couplings, only 15 couplings could be measured with confidence. However, these couplings span residues from the various secondary structure elements of the protein and this is adequate to test the validity of using the crystal structure to approximate the geometry in solution. The order tensor determined for MBP, using measured RDC values from amide 1H – 15N pairs and the crystal structure of the trimeric form (PDB entry 1RTM) is shown in Figure 5. As can be seen from the Figure, the position of the direction of highest order (red dots labeled Szz ) coincides with the 3-fold symmetry axis as seen from a coordinate frame that uses the crystal structure 3-fold axis as the z-axis. The other principal axes of the order tensor are shown as blue and black dots. These ring the principal axis with no definitive depiction of directions for Sxx and Syy within this ring. This pattern is characteristic of a system showing axial symmetry. It confirms the axially symmetric nature of ordering of MBP, and the position of the principal order axis rules out the possibility of a CRD being displaced from the orientation seen in the crystal structure. As a second test of consistency of crystal structure geometry with geometry in solution, we plot in Figure 6 experimental values for dipolar couplings against predicted values from the crystal structure geometry and the best-fit order tensor parameters. The slope close to 1 and the modest scatter (correlation coefficient of 0.95) suggest that the crystal structure is a good representation of the situation in solution, thereby ruling it out as a likely source for the observed deviation in the binding geometry of AMM.

Figure 6. Correlation plot of experimentally measured RDCs versus predicted RDCs from the crystal structure of MBP trimer.

456

Mannose-binding Protein –Trimannoside Complex

Figure 7. (a) Trimannoside ligand used for binding studies with MBP. Man I, Man II and Man III represent rings 1, 2 and 3 of trimannoside, respectively, while f; c; v represent the glycosidic dihedral angles. (b) Section of a 1H– 13C coupled HSQC spectra of trimannoside showing 1H – 13C couplings in the anomeric region of the three mannose units in the free state and (c) in complex with trimeric MBP. Spectra were recorded for the two samples in isotropic phase and in aligned phase (10% (w/v) bicelle solution). RDCs were calculated as the difference between the coupling values in isotropic and aligned phase shown on the spectra.

Measurement of RDCs and order tensor determination for trimannoside bound to MBP For a ligand with sufficiently rigid geometry, dipolar couplings represent a powerful way of angularly constraining the ligand relative to the protein to which it binds. Dipolar couplings have been applied before in obtaining the ligand geometry of AMM bound to MBP.9 We have used similar methods in this study to obtain orientational constraints from dipolar couplings for the binding of trimannoside to MBP, but we have not used the isotopic enrichment found necessary in the previous study and have extended the study

to a much larger ligand. For the binding studies, a trimeric form of MBP was used. RDCs for trimannoside in the free state and complexed with MBP were measured from one bond 1H – 13C J-couplings in the proton dimension of 1H – 13C HSQC spectra acquired without proton decoupling (Figure 7(b) and (c)). Two sets of spectra were acquired for samples dissolved in bicelle media; one in isotropic phase and the other in aligned phase corresponding to bicelles at 25 8C and at 35 8C, respectively. RDCs were calculated as the difference in couplings measured for the isotropic phase and aligned phase and are listed in Table 1. An eight times molar excess of ligand over the protein was used

457

Mannose-binding Protein –Trimannoside Complex

Table 1. RDC measurements for free and bound trimannoside Free trimannoside 1H– 13C coupling (Hz)a

RI C1 – H1 RI C2 – H2 RI C3 – H3 RII C1 –H1 RII C2 –H2 RII C3 –H3 RIII C1 –H1 RIII C2 –H2 RIII C3 –H3

Bound trimannoside 1H– 13C coupling (Hz)a

Isotropic

Aligned

RDCb

Isotropic

Aligned

RDCb

Bound RDCc

171.2 149.4 143.0 171.0 149.4 143.5 173.8 149.4 143.7

181.3 142.9 143.4 174.3 153.3 130.5 189.5 130.2 157.3

10.1 26.5 0.4 3.3 3.9 213.0 15.7 219.2 13.6

171.2 149.4 143.0 171.0 149.4 143.5 173.7 148.9 143.7

180.2 143.7 142.6 274.0 153.4 131.9 187.1 131.9 154.5

9.0 25.7 20.4 3.0 4.0 211.6 13.3 217.0 10.8

24 4 28 22 5 5 214 28 220

a 1

H– 13C couplings were measured with a precision of ^0.3 Hz. RDCs for free and bound trimmanoside were calculated with a precision of ^0.5 Hz. c Bound state RDCs were calculated using equation (1). Extrapolated values for the bound state have an estimated precision of approximately 5 Hz. b

to increase the signal-to-noise ratio of the observed ligand signals. Since this ligand undergoes rapid exchange on and off the protein, RDCs are observed as an average between the free and bound states of trimannoside, with the bound state trimannoside corresponding to approximately 8%. Even with some preferential orientation of the protein complex, the contribution from the free state dominates the observed RDCs. However, RDCs for the complexed sample are different enough from those observed in the free trimannoside to extract bound state couplings using known equilibrium constants. This process requires good signal-to-noise and resolution in the acquired spectra, as observed in our trimannoside spectra at 800 MHz. In future, the availability of higher magnetic fields and cryoprobes will further facilitate this process. The order tensor for trimannoside in the free state and in the bound state was calculated using the RDCs in Table 1, and the geometry of free and bound trimannoside determined from modeling studies by Sayers (vide infra).20,32 As can be seen in Figure 8, unlike the order tensor for free trimannoside, the order tensor for bound trimannoside is axially symmetric and reflects the axially symmetric order tensor of MBP. With the experimentally determined order tensor for bound trimannoside in hand, the orientation of trimannoside relative to the symmetry axis of MBP was obtained by rotating the molecular frame of trimannoside to align the order tensor of bound

trimannoside with that of MBP. Once aligned, it was translated and rotated about the symmetry axis to direct the O3-linked mannose toward the binding site Ca2þ as suggested in the work by Sayers20 using the program MolMol.33 This relative orientation was subsequently used as a starting point for the docking studies. Docking of trimannoside to MBP No crystal structure for trimannoside bound to MBP-A exists, although several structures of different oligosaccharides complexed to the CRD of both dimeric and trimeric MBP are available.34 Recently, Sayers et al. presented a model for the conformation of trimannoside, free and in complex with an MBP dimer, using NMR and molecular dynamics methods.20,32 The bound-state geometry for trimannoside was calculated using transfer NOE data input as distance constraints in a simulated annealing protocol. Several different conformations for bound trimannoside were computed, corresponding to different values for the torsional angles spanning the a(1 ! 3) and a(1 ! 6) linkages, and modeled into the binding site of MBP using molecular dynamics. A conformation corresponding to v ¼ 608; f ¼ 648; w ¼ 1808 for the a(1 ! 2 . 6) linkage and f ¼ 858; w ¼ 21058 for the a(1 ! 3) linkage was found to be one of the most populated during the simulations. This conformation was used as the starting ligand geometry for our docking studies as well as the order

Figure 8. Sauson – Flaumsteed projection of the directions of highest order for oriented trimannoside (a) in the free state and (b) bound to MBP. The continuous band in the latter plot reflects the axial symmetry of MBP. x; y; z represent the molecular coordinate frame of the trimannoside model in each case.

458

Figure 9. Structure of the trimannoside – MBP complex resulting from the docking of oriented trimannoside to MBP. A single CRD of the trimeric MBP is shown for clarity. Calcium atoms are shown in green; Man I, Man II and Man III represent rings I, II and III of trimannoside, respectively. The Figure was generated using the molecular visualization program RASMOL.

tensor calculation for both free and bound trimannoside described above. The program AutoDock35 was then allowed to search for the best binding geometry for trimannoside in the binding site, allowing only a minimal change in the torsional angle values (^ 208) for trimannoside using hard torsional constraints. The protein was modeled as a rigid body with no change in any of the torsional angles of the protein. The orientation of the ligand relative to the protein was not constrained during the search. The AutoDock search produced a minimal deviation from the orientation determined from RDC measurements (symmetry axis deviation , 208). All the torsional angles in the docked trimannoside retained values similar to those of the starting structure with small deviations (, 108). The trimannoside-docked structure presented in Figure 9 shows contacts with the calcium and other residues in the binding site of MBP similar to those observed in the modeled structure presented by Sayers et al.20 Specifically, the 3-OH and 4-OH groups of the terminal mannose molecule are within the coordination shell of calcium and oriented in a manner that puts the 3-OH within hydrogen-bonding distances of Glu185 and Asn187, while the 4-OH is in close proximity to Asn205 and Glu193. This corresponds to an orientation (orientation I) observed for other oligosaccharide ligands binding to MBP-A as reported by Weis and co-workers.21 It is similar in orientation and internal structure to the trimannoside terminus of the oligosaccaride in the original crystal structure of MBP-A, with the two structures ˚. overlaying to an rmsd , 3 A

Mannose-binding Protein –Trimannoside Complex

While the experimental determination of the direction for a single symmetry axis does not by itself allow definition of ligand-binding geometry, the utility of RDC data is quite apparent when used in combination with modeling and other experimental data. The acquisition of RDC data is efficient and provides a method for determining relative orientation of ligand to the protein without extensive assignment of protein resonances or collection of ligand – protein NOEs. In the case of homomultimeric proteins with appropriate symmetry properties it may be possible to omit the collection of NMR data on the protein entirely. The data presented here show that the MBP trimer exhibits axial symmetric ordering with the symmetry axis aligned with the 3-fold axis and the crystal structure supports that contention. The study does not, however, resolve the discrepancy in sugar orientation noted between our original study of AMM bound to MBP-A in solution and the crystal structure of MBP-C containing AMM. In the context of this study, an interesting observation has been highlighted by Weis and co-workers.21 Although the vicinal O3 and O4 oxygen atoms are always involved in coordination to the active Ca2þ, the position of these oxygen atoms in the coordination shell is reversed in MBP-A and MBP-C. Thus, there is a nearly 1808 rotation in the ligand when comparing the AMM orientation in MBP-C and that of the terminal mannose on the oligosaccharide in MBP-A. This obviously raises questions about whether the original comparison to the MBP-C crystal structure was appropriate. Weis and co-workers have now supplied a structure of AMM in MBP-A.21 This actually mimics the O3, O4 order seen in MBP-C; i.e. there is no reversal in AMM orientation between MBP-C and MBP-A structures. However, given that the orientation is slightly different, we have now examined the fit of the original AMM data to the new AMM-MBP-A crystal structure by back-calculating RDCs from the structure (data not shown). The fit is well outside experimental error. Hence, the structure with the 408 deviation in the orientation of AMM with respect to the symmetry axis of MBP remains the best solution model. The new data on variation in orientations of saccharides bound to MBP-A in the recent studies may still offer an explanation for the differences seen for AMM bound to MBP-A in solution and in crystals. The studies do suggest that there is considerable variation in allowed binding geometry. In fact, crystal structures of MBP-A with other monosaccharide and disaccharide ligands have been observed with mixtures of binding modes. Relatively small energetic differences can easily shift populations from one of these modes to another. As noted by Weis and co-workers, the bound orientation is determined by the nature of the glycoside and the kind of intermolecular contacts it makes with residues in the binding site of MBP-A. In the case of AMM, with a methyl group

459

Mannose-binding Protein –Trimannoside Complex

as opposed to the larger sugar molecule that is normally present at the glycosidic oxygen atom, subtle adjustments in the protein-binding site may occur, and some of these may be allowed in solution but not in the crystal. It is likely that our observed deviations result from a combination of these subtle adjustments and some motional averaging within the binding site. Although asymmetric averaging would cause departures from apparent axial symmetry, these may be hard to see with the limited amount of data available for AMM. It is well known that the actual positions of x and y order axes are hard to determine in any case.23 For our trimannoside, difficulties in determination are likely to be less. First, movement in the binding sites is likely to be restricted by interactions between protein and sugars other than the terminal mannose. Second, the number of RDCs measured is larger. The excellent agreement between structures in this case is encouraging. The demonstrated ability to collect data without the need for 13C enrichment is also a promising feature. This means that possibilities for investigation of a number of related ligands can be pursued in the future.

Materials and Methods Expression and purification of MBP For expression of MBP, the E. coli strain BL-21-CodonPluse-RIL (Stratagene) containing extra copies of the E. coli arg U, ile Y and leu W tRNA genes was used. The tRNAs encoded by these genes recognize the rare AGA/AGG codons for arg that are present in the plasmid containing the MBP gene. Usage of this strain allowed a higher level of protein expression compared to conventional BL-21 strains. The following protocol was utilized for expression of isotopically labeled protein—BL-21-CodonPlus cells transformed with plasmid containing the MBP gene were used to inoculate 50 ml of LB medium for initiation of cell growth. After overnight growth at 37 8C, the cells were spun down and transferred to 1 l of defined medium containing M9 minimal media salts (6.5 g of anhydrous Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl), 1 mM MgSO4, 0.1 mM CaCl2, 0.002% (w/v) thiamine and 1 ml of a micronutrient solution containing trace metal elements. For uniform labeling of the protein with 15N, 1 g of 15NH4Cl was added to this medium along with 2 g of unlabeled glucose. Deuteration of the protein was achieved by growing the cells in partially deuterated defined medium (75% (v/v) 2H2O, 25% (v/v) H2O). This allowed for 50% random fractional deuteration of MBP, as confirmed by mass spectroscopy. For uniform 13C, 15N labeling, 1 g of 15NH4Cl and 2 g of 13C6-glucose was added to the above medium. The cells were grown further in this defined medium till they reached an absorbance of , 1.0 at 600 nm. At this point they were induced for protein expression with 1 ml of 0.5 M isopropyl-b,D -thiogalactopyranoside. They were then grown for an additional three hours and harvested. For purification, a modified protocol different from other published purification protocols for MBP was used.26 Harvested cells were resuspended in a lysis buffer containing 25 mM Tris –

HCl (pH 7.8), 10% (v/v) glycerol and 150 mM NaCl. Lysis was accomplished using the French Press. Following lysis, urea was added to the lysed solution to a final concentration of 8 M. After stirring for about half an hour, b-mercaptoethanol was added to the solution to a concentration of 0.1% (v/v) and the solution was stirred at 4 8C for about an hour. This resulted in complete denaturation of the protein. After spinning down in an ultracentrifuge (108,000 g for one hour at 0 8C) to remove cell debris, the solution was diluted eightfold with load buffer (25 mM Tris – HCl (pH 7.8), 25 mM CaCl2, 1.25 M NaCl) and stirred overnight at 4 8C to refold the protein. The solution was then loaded onto a mannose-Sepharose affinity column for purification. MBP binds to the affinity column, while the other proteins are removed by washing with load buffer. MBP was eluted from the column by using an elution buffer (25 mM Tris –HCl (pH 7.8), 2.5 mM EDTA, 1.25 M NaCl). Fractions containing MBP were pooled, concentrated and dialysed against NMR sample buffer (25 mM Tris – HCl (pH 7.8), 25 mM CaCl2, 50 mM NaCl) before being used for NMR. Concentrations of the NMR samples used ranged from 0.7 mM to 1 mM (monomeric concentrations). Preparation of the dimeric form of MBP A dimeric form of MBP was prepared by proteolytic digestion of the MBP trimer with trypsin as described.26 Specifically, 1 ml of 1 mM MBP trimer was incubated at 37 8C with trypsin (25 ug/ml) for one hour. The mixture was then loaded onto a mannose-Sepharose column and the dimeric form purified in a manner similar to that described above for the trimer and further confirmed by native gel electrophoresis. This MBP dimer is devoid of the coiled-coil helical region and consists only of the C-terminal CRD (residues 107– 221) with an effective molecular mass of 26 kDa.26 NMR spectroscopy All NMR experiments were performed on a Varian Inova 800 MHz spectrometer unless noted otherwise. Backbone assignments were carried out for the dimeric form of MBP using a 13C,15N uniformly labeled sample (1 mM). Sequential connectivities were obtained from two standard 3D experiments, namely HNCA and HNCACB. HNCA data were collected using a constanttime version of the 3D HNCA experiment36 and consisted of 1024 £ 64 £ 32 complex data points for the 1H, 13 C and 15N dimensions, respectively. HNCACB data were collected using a 3D HNCACB experiment36 and consisted of 1024 £ 64 £ 24 complex data points for the 1 H, 13C and 15N dimensions, respectively. Quadrature detection in indirect dimensions was achieved by hypercomplex data acquisition. For measurement of RDCs, a 0.7 mM sample of the MBP trimer was used. 1H– 15N couplings were measured using a coupling enhanced version of a 1H– 15N TROSY experiment (Figure 3). In the CE-TROSY experiment, an extra coupling evolution period added after the t1 evolution period is incremented in an accordion fashion. The incrementation of the coupling evolution period is controlled by the parameter k. Two sets of experiments were carried out for coupling measurements on the trimer. In the first set, data were collected for a sample of MBP in a 6% (w/v) bicelle solution (1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-snglycero-3-phosphocholine (DHPC), 3:1, w/w) in isotropic

460

Mannose-binding Protein –Trimannoside Complex

phase at 25 8C and in the second set, data were collected with the same sample corresponding to the aligned phase of bicelles at 35 8C. For each CE-TROSY experiment performed, two data sets were collected, one corresponding to a value of k ¼ 0 and the other where k ¼ 0.7 (for the isotropic sample) and k ¼ 1 (for the aligned sample). The difference in positions of peaks in the two data sets corresponds to a fraction of the value of JNH. The isotropic and aligned phase couplings were scaled depending on the value of k to correspond to the value of JNH and dipolar couplings calculated as difference in values of these scaled couplings. Trimannoside used in the binding studies with MBP was obtained from BIOMOL Research Labs, Inc. For the NMR experiments, two samples were prepared. The first sample consisted of 4 mM trimannoside, 10 mM NaCl, 10 mM Tris – HCl (pH 7.8), 25 mM CaCl2, 95% 2 H2O in 10% (w/v) bicelle solution. The second sample consisted of 0.25 mM MBP trimer, 2 mM trimannoside, 10 mM NaCl, 10 mM Tris – HCl (pH 7.8), 25 mM CaCl2, 95% 2H2O in 10% (w/v) bicelle solution. 1H – 13C couplings were measured at natural abundance for trimannoside in each sample using a coupled 13C HSQC spectrum where the decoupler was turned off during acquisition. Two sets of data were collected for each sample corresponding to the bicelle solution in isotropic phase (25 8C) and aligned phase (35 8C). RDCs were calculated as the difference in couplings between the isotropic and aligned phase. All sets of data were processed and analyzed using the Felix software package (Molecular Simulations).

Calculation of order tensors Dipolar couplings measured for trimannoside correspond to an average between the free state and the bound state of trimannoside. Bound-state dipolar couplings for trimannoside were calculated from the equation: Db ¼ ðDobs 2 xf Df Þ=xb

Docking studies For docking of trimannoside to MBP, the program AutoDock 3.05 was utilized.35 The crystal structure of the MBP trimer was used as a starting structure for docking trimannoside.22 Since trimannoside binds independently and equivalently to each monomer in the trimer, coordinates for only one monomer were used for the docking. All water molecules were removed from the structure. Polar hydrogen atoms were added to the protein using the Autodock utility protonate, while the non-polar hydrogen atoms were treated under a united-atom approach. KOLLUA partial charges and atomic solvation parameters were generated for the protein using the AutoDock utilities q.kollua and addsol, respectively. The molecular modeling program Chem3D (CambridgeSoft) was used to build a starting structure for the trimannoside ligand. Torsional angle values for the rotatable bonds in trimannoside, namely f; c for the a(1 ! 3) linkage were fixed at 858; 21058; respectively, while the values for f; c; v for the a(1 ! 6) linkage were fixed at 648; 1808; 608; respectively. All polar and non-polar hydrogen atoms were included in the starting structure. Partial charges were assigned to the ligand using q.kollua. The torsional values for rotatable bonds in trimannoside described above were allowed to move in a range of ^208 by applying hard torsional constraints during the docking process. Mass-centered Grid maps were built using the program AutoGrid with ˚. 60 £ 60 £ 60 points and a grid spacing of 0.3 A Lennard – Jones 12-10 and 12-6 parameters included in the program were used for modeling the van der Waals interactions and hydrogen bonds, respectively. The docking was performed by AutoDock using a simulated annealing protocol with a total of 50 runs with step ˚ for translation and 58 for torsion. The sizes of 0.2 A rmsd tolerance for docked ligand clusters was set at ˚ . The docked ligand structures were extracted with 1A the get-docked utility included in the program and analyzed using the graphical molecule-viewing program MolMol.33

ð1Þ

where Db is the dipolar coupling for trimannoside in the bound state, Dobs is the observed dipolar coupling, xf is the fraction of trimannoside in the free state, Df is the dipolar coupling measured for trimannoside in the free state and xb is the fraction of trimannoside in the bound state. The fraction free and fraction bound for trimannoside were calculated using the known association constant of trimannoside to MBP corresponding to a value of 0.94 mM21.20 Order tensors were determined using the calculated bound-state dipolar couplings and associated coordinates that were input into a singular value decomposition program for the determination of order tensor elements.37 For the order tensor determination of MBP, a total of 15 RDC values were used. These values correspond to residues for which assignments are available and there is no significant overlap of resonances or change in chemical shift positions between the trimeric and the dimeric forms. Except for the bound trimannoside, where estimated experimental errors were used, errors in dipolar couplings were set to approximately twice the estimated experimental precision to allow for imperfections in structural representations.

Acknowledgements This work was supported by a grant from the National Institutes of Health (GM33225). The authors thank Ziad Eletr for help with purification of MBP-A, Dr Hashim-Al Hashimi for useful discussions, and Dr K. Drickamer for providing the initial MBP-A gene.

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Edited by M. F. Summers

(Received 5 November 2002; received in revised form 10 February 2003; accepted 10 February 2003)

Supplementary Material for this paper comprising two Tables is available on Science Direct