Structures of two <i>Arabidopsis thaliana</i> major latex proteins represent novel helix‐grip folds

Abstract

The major latex proteins (MLPs) are a protein family first identified in the latex of opium poppy (Papaver somniferum).1 They are found only in plants and have 24 identified members in Arabidopsis alone as well as in other plants such as peach, strawberry, melon, cucumber, and soybean. While the function of the MLPs is unknown, they have been associated with fruit and flower development and in pathogen defense responses. Based on modest sequence similarity, they have been characterized as members of the Bet v 1 protein superfamily2; however, no MLP structures have yet been reported. Additional sequence comparisons have expanded the Bet v 1 superfamily to include the (S)-norcoclaurine synthases and cytokinin-specific binding proteins (CSBPs) as well as the MLP and intracellular pathogenesis-related class 10 (PR-10) families.3, 4 PR-10 proteins, which include tree pollen allergens and major food allergens, are expressed in various tissues and organs in response to pathogen attack as well as environmental stresses such as drought, wounding, ultraviolet radiation, and oxidative stress. The Bet v 1 superfamily is further classified as a member of the Pfam clan, Bet v 1-like (CL0209), which includes the following diverse families outside the plant kingdom: ASHA1, COXG, IP trans, Polyketide cyc, Ring hydroxyl A, and steroidogenic acute regulatory-related lipid transfer (START). More than 100 Bet v 1 superfamily sequences have now been identified,5 and three-dimensional structures have been determined for at least 12 of these proteins: birch pollen allergen Bet v 1,6 Bet v 1l, a hypoallergenic isoform of Bet v 1,7 major cherry allergen Pru av 1,8 two proteins from yellow lupine subclass LIPR-10.1,9 two from subclass LIPR-10.2,10, 11 Pachyrrhizus erosus (jicama) SPE-16 (PDB code 1TW0), major celery allergen Api g 1,12 CSBP from mung bean,13, 14 and norcoclaurine synthase from Thalictrum flavum (2VNE, 2VQ5). The Bet v 1 fold consists of a curved seven-stranded β-sheet wrapped around a long C-terminal helix, α3, and has been classified as a type of “helix-grip” fold.15 Between these two structural elements is a large, Y-shaped hydrophobic cavity, which is closed at one end by two short helices, α1 and α2, that connect strands β1 and β2. The role of this hydrophobic cavity as a ligand-binding pocket was first suggested based on structural similarities between the hydrophobic cavity of Bet v 1 and the cholesterol-binding pocket of the START domain of the human protein MLN64.15, 16 Based on this hypothesis, NMR titration experiments of Pru av 1 binding to homocastasterone were conducted, which provided the first evidence that this cavity could bind plant steroids.8 More recently, structures of Bet v 1l in complex with deoxycholate,7 a compound structurally similar to the plant hormones, brassinosteroids, and CSBP in complex with the cytokinin, zeatin,13 enabled the determination of specific binding interactions within the hydrophobic pockets. In both cases, the hydrophobic binding pocket could accommodate two ligand molecules. In addition to the hydrophobic pocket, the Bet v 1 fold is characterized by a rigid and highly conserved glycine-rich loop, or so-called “P-loop” motif, which has the sequence GxGGxGT. In studies of the complex between Bet v 1 and an IgG Fab′ fragment from mouse (human IgE mAbs are difficult to obtain in sufficient quantities from allergic patients), the P-loop sequence was shown to be contained within the binding surface, suggesting that its rigid conformation helps define the IgE epitope.17, 18 Here we report the first structures of two MLP proteins, which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (SwissProt/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as fold-space targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17–173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobic binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general. Phylogenetic tree showing the relationships between representative members of the major latex protein (MLP) family. A protein construct corresponding to residues 17–173 of A. thaliana gene At1g70830.1 (MLP28) was prepared in [U-13C,15N]-labeled form according to CESG wheat germ cell-free protocols as previously described.21 Briefly, the protein was expressed with an N-terminal His6 fusion tag in wheat germ extract supplemented with [U-13C,15N] amino acids (Cambridge Isotope Labs) and purified by Ni-NTA affinity chromatography, followed by size-exclusion chromatography. At1g24000.1 was prepared by heterologous expression in E. coli as previously described.22, 23 The sample used for NMR contained ∼0.7 mM [U-13C,15N] MLP28, 10 mM deuterated bis-Tris (pH 7.0), and 5 mM dithiothreitol in 95% H2O and 5% 2H2O. All NMR data were acquired at 25°C on a Bruker 600 MHz spectrometer equipped with a triple-resonance CryoProbe™ and processed with NMR Pipe software. Chemical shift assignments and 3D structure determination of MLP28, and chemical shift mapping of progesterone binding to At1g24000.1 were performed as detailed in the Supporting Information Methods. The At1g24000.1 protein was subjected to the UW192 crystallization screen and the structure was solved using single-wavelength anomalous diffraction (SAD) methodology as described in the Supporting Information Methods. Coordinates and restraints have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.pdb.org/), under PDB code 2I9Y (MLP28) and PDB code 1VJH (At1g24000.1). All time-domain NMR data and chemical shift assignments have been deposited in BioMagResBank (http://www.bmrb.wisc.edu/) under BMRB accession number 15720 for MLP28. The sequence of MLP28 consists of two domains which share 93% sequence identity with each other.24 Only one other A. thaliana MLP family member, MLP34, contains two duplicated domains. This gene is considered to be a paralog of MLP28, along with three other genes, predicted to have arisen from tandem duplications within the A. thaliana genome in an analysis of phylogenetic relationships and genomic positional data.24 We determined the solution structure of one domain of MLP28 (referred to herein simply as MLP28), corresponding to residues 17–173, after unsuccessful attempts to crystallize the entire protein. Under the solvent conditions used, MLP28 was a stable, folded monomer. However, increased ionic strength led to a tendency for the protein to aggregate into higher order oligomers (data not shown). An automated procedure for iterative nuclear Overhauser effect (NOE) assignment25 was used to calculate the structure, and molecular dynamics calculations in explicit solvent were used in the final refinement.26 The final conformers were generated from 1766 nonredundant NOE-derived distance restraints and 197 dihedral angle constraints (Supporting Information Table 1). Structure determination efforts on full-length At1g24000.1 were conducted in parallel using both NMR and X-ray crystallography. Nearly complete 1H, 15N, and 13C resonance assignments were determined,27 but further work on the solution structure was abandoned once the crystal structure was solved. The At1g24000.1 crystal belongs to the space group P21 with unit cell parameters of a = 45.68 Å, b = 34.26 Å, c = 78.56 Å, α = 90.00°, β = 90.01°, and γ = 90.00°. The asymmetric unit contains two protein molecules. It is unknown whether this dimer is biologically relevant. The MLP28 and At1g24000.1 structures both display the helix-grip fold,15 albeit with significant differences from the typical Bet v 1 fold. The MLP28 structure consists of a six-stranded β-sheet enfolding a 21-amino acid C-terminal α-helix [Fig. 2(A,B)]. A short α-helix is positioned at one end of a large hydrophobic cavity and a single turn of 310 helix, in the loop between strands β4 and β5, is found at the other end. (A) Structure of the MLP28 domain. Backbone trace of the ensemble of 20 NMR structures is shown as a stereo image, with α-helices in violet and β-strands in green, produced using the program PyMOL.28 (B) Ribbon diagram of the lowest energy MLP28 conformer. (C) Backbone rmsd values and 1H-15N heteronuclear NOEs plotted for each residue of MLP28. (D) Ribbon diagram of At1g24000.1 (E) Superposition of MLP28 (green) and At1g24000.1 (orange). The MLP28 structure deviates significantly from the conserved Bet v 1 fold in that the β2 strand and the second short α-helix between strands β1 and β2 of the Bet v 1 fold are replaced by a long, flexible loop comprising residues 45–65. Heteronuclear NOE values of less than 0.6 for residues 45–60 confirm that the loop is mobile and highly unstructured [Fig. 2(C)]. The amide resonances of residues 62–65 were undetected; therefore, these 15N-1H NOE values could not be determined. The glycine-rich loop (residues 66–72), which is adjacent to the unstructured loop, is somewhat rigid as indicated by 15N-1H NOE values of 0.6–0.8 and lower rmsd values than residues 45–65. Strands β2 and β3 (residues 76–96), which stretch out across the top of the molecule perpendicular to the C-terminal helix, display higher rmsd values. However, 15N-1H NOE values similar to the rest of the molecule suggest that the strands fluctuate on a slower timescale (millisecond to microsecond) as a rigid body. A potential salt bridge between His81 (in the loop between β2 and β3) and Glu160 (in the long α-helix) may serve to stabilize the strands, which serve as a lid over the largest hydrophobic cavity opening. In the NMR structure of Pru av 1, the equivalent loop is much shorter and shows significant internal flexibility as indicated by low 15N-1H NOE values.8 This loop is also poorly defined in both the solution and crystal structures of Bet v 1.6 In contrast, the crystal structure of yellow lupine LlPR-10.2B in complex with zeatin contains a salt bridge in the same position as that of MLP28, which similarly serves to “gate” the cavity opening.11 The At1g24000.1 crystal structure displays a fold similar to that of MLP28, consisting of a five-stranded β-sheet and a 21-amino acid C-terminal α-helix [Fig. 2(D)]. Like MLP28, it also has a single short α-helix (α1) and an equivalent 310 helix. The two structures can be superimposed with an rmsd of 1.6 Å for 102 aligned Cα atoms [Fig. 2(E)]. However, as noted earlier, the At1g24000.1 structure differs significantly from MLP28, as well as the Bet v 1 fold, because of the deletion of 33 amino acids following the α1 helix. Compared with MLP28, the majority of the flexible loop, the glycine-rich loop, and the β2 strand are absent. As a result, the hydrophobic cavity is noticeably smaller than that of MLP28. Also, there is only a small loop between α1 and the equivalent β3 strand, rather than a long flap covering the cavity. Additional electron density observed in the cavity was ascribed to low-molecular-weight polyethylene glycol. A search for structures homologous to MLP28 using the Secondary Structure Matching (SSM) server (http://www.ebi.ac.uk/msd-srv/ssm/29) yielded At1g24000.1 as the top match (Q-score = 0.43). Alignment statistics for the next closest structural neighbors, CSBP from mung bean in complex with zeatin, SPE-16, yellow lupine LlPR10.1B and L1PR10.2B, and Bet v 1 are summarized in Supporting Information Table 3, and representative homologs are depicted in Figure 3(A). MLP28 and At1g24000.1 display low sequence homology to the most similar Bet v 1 superfamily structures (20% identity or less). A structure-based sequence alignment of representative MLPs and the homologous structures highlight some interesting sequence differences [Fig. 3(B)]. In particular, a conserved cluster of residues is found at the closed end of the hydrophobic pocket consisting of Tyr115, Lys116, Ser117, Phe118, Glu142, and Lys143 (MLP28 numbering), which is not conserved in the PR-10 structures. CSBP differs from the other two structures in that Ser117 is conserved and Phe118, Glu142, and Lys143 are conservatively substituted with Tyr, Asp, and His, respectively. A number of other residues in the MLP28 cavity, such as Trp77, Glu90, Ile102, and Phe118, are conserved or conservatively substituted in CSBP but not in others. Notably, the residues in CSBP corresponding to Trp77 and Glu90 are ligand-binding residues. (A) Ribbon diagrams of MLP28, At1g24000.1, and representative structural homologs CSBP from mung bean (2FLH), birch pollen allergen Bet v 1 (1BV1), and jicama SPE-16 (1TW0). (B) Sequence alignment of selected MLP family proteins as well as the three structural homologs shown in panel A. Strictly conserved residues in >70% of the sequences are highlighted in black and those that are conservatively substituted are highlighted in gray. Stars denote residues of CSBP that interact with the ligands via hydrogen bonds, and “+” denotes residues that interact via van der Waals interactions. These patterns of sequence conservation suggest that the MLPs are more closely related to CSBP than the other PR-10 proteins. It is interesting, however, that none of these four residues are strictly conserved in At1g24000.1. Additional residues that are conserved in the MLPs, including At1g24000.1, are found just before and within the long α helix (Pro151, Leu155, and Ile165). The proline likely serves a structural role in determining the turn of the main chain and thereby positioning the helix within the groove of the β-sheet. Conservation of the two hydrophobic residues appears to be significant, especially because the helix is the most variable region, both in structure and sequence, among the PR-10 proteins. This suggests that these residues are important in determining the binding specificity of the MLPs. Another key sequence difference between the MLPs and the other Bet v 1 proteins is the lack of conservation of the glycine-rich loop (GxGGxGT). In all the MLPs, except At1g24000.1 in which the loop is absent, this sequence is GxxxxxG, with the third amino acid of the motif usually Trp. Since the MLPs have no known allergenicity, this loop is not expected to serve as a IgE epitope, as with the PR-10 proteins.19 However, it is interesting that the loop has retained its rigid conformation. It remains to be determined whether the modified loop serves a functional role. The ability of At1g24000.1 to bind a hydrophobic ligand was investigated by an HSQC titration of [U-13C,15N] At1g24000.1 with progesterone. While progesterone is a human hormone not found in plants, it shares enough structural similarity to the plant steroid, brassinolide, to serve as a representative ligand. Combined 1H/15N chemical shift perturbations are shown in Figure 4(A). Significant changes (>0.4 ppm) are clustered in three regions, Ala23-Glu41, Ile52-Ile59, and Ile102-Leu113 [Fig. 4(B)]. It can be seen that the affected residues are located at the opening as well as in the bottom of the cavity, providing evidence that this cavity can accommodate a steroid molecule. Included within these regions are the conserved hydrophobic residues of the long α helix, which were identified above as potential hydrophobic binding ligands on the basis of sequence conservation among the MLPs. It is therefore likely that the cavity in MLP28 and other MLPs can bind a ligand in a similar manner, but further studies are needed to determine the specific mode of binding and whether more than one ligand can bind in the pocket. (A) Combined 1H/15N chemical shift perturbations for each backbone amide group of At1g24000.1 upon binding of 5 mM progesterone plotted as a function of At1g24000.1 residue. Residues which displayed perturbations higher than 0.4 ppm are colored green. Along with the Pro residues, the amides of Ser1, Thr2, Thr28, Lys34, Thr38, His100, and Ser101 were unassigned. (B) The residues showing the highest chemical shift perturbations upon progesterone binding are highlighted on the structure of At1g24000.1, shown in the same orientation as in Figure 2(D) and after a 180° rotation about the vertical axis. Although the function of the MLPs is unknown, gene expression of A. thaliana MLPs has been shown to be affected by a variety of environmental stimuli or stresses. One such stimulus is gravity, which has a pronounced effect on plant root growth. A study involving whole-genome microarray analysis of Arabidopsis root apices after gravity and mechanical stimulation showed that transcription of the At1g70830 (MLP28) gene, as well as the highly similar At1g70890 (MLP43) gene, was upregulated within 15 min of gravity stimulation. Another MLP (At4g23670) gene was among a cluster of five genes upregulated by more than threefold within 2 min. All three of these genes were then subsequently downregulated below control levels within 60 min.30 Based on the rapid and transient response to the stimulus, MLP gene expression can be considered a specific response to the gravity stimulation, rather than being a secondary response. It is possible that these MLPs play a role in gravitropism, the response of a plant to gravity, through binding of hormones in the hydrophobic pocket. The hormone auxin, in conjunction with brassinosteroids, is known to play a central role in gravitropism31, 32 along with other plant hormones such as cytokinin, ethylene, and gibberellic acid.30 Therefore, there is a wide range of putative MLP binding ligands. In addition to their role in gravitropism, the MLPs are differentially expressed during plant development. For example, At4g23670 is expressed during and development only and is found only in the the other expression of which shows sequence identity, development and is found in all tissues such as and MLP28 shares the highest sequence homology with peach and cucumber both of which are highly expressed during fruit is the only plant known to both an MLP and PR-10 protein For this it that the functional role of MLPs be similar to that of the PR-10 proteins. It is likely that the MLPs out their function in plant and development through the binding of hormones or other in a conserved hydrophobic cavity. The first two structures solved for members of the MLP family a helix-grip fold that differs significantly from the Bet v 1 fold. low sequence MLP28 and At1g24000.1 are structurally similar and share a number of unique conserved residues when compared to other Bet v 1 superfamily proteins. However, the deletion of 33 residues in the At1g24000.1 sequence in a more structure with a small hydrophobic cavity. the structural we that At1g24000.1 is to bind a representative progesterone, that At1g24000.1, and in all the MLP can in a wide variety of through plant cell The are to of the and Center for cluster of structure software. of the was by the of of of the 14 was by the of Center for Research Additional Supporting Information may be found in the of this The is not for the or of by the than be to the corresponding for the