NMR Structure of the Peptidyl-tRNA Hydrolase Domain from Pseudomonas syringae Expands the Structural Coverage of the Hydrolysis Domains of Class 1 Peptide Chain Release Factors

Abstract

The NMR structure of the peptidyl-tRNA hydrolase (PTH) domain from Pseudomonas syringae (P. syringae PTH domain) was solved by using recently devised protocol for high throughput protein structure determination. The P. syringae PTH domain belongs to a large Pfam family PF00472, which consists of at least 1549 proteins annotated as ‘hydrolysis domains of peptidyl-tRNA’. The structure of P. syringae PTH domain expands the ‘structural coverage’ of the PFam family.

Introduction

The 100-residue peptidyl-tRNA hydrolase domain from the gram-negative bacterium Pseudomonas syringae infecting tomato, referred to in this paper as the ‘P. syringae PTH domain’, (gi|42602314, SwissProt/TrEMBL ID Q885L4_PSESM, access number Q885L4)¹ constitutes the hydrolysis domain of peptide chain release factor (RF) encoded by gene PSPTO1818. The P. syringae PTH domain has no significant amino acid sequence similarity to any protein with known three-dimensional structure, and was selected by the Protein Structure Initiative-2 of the United States National Institutes of Health and assigned to the Northeast Structural Genomics consortium (NESG; http://www.nesg.org) (NESG target ID PsR211). The P. syringae PTH domain belongs to the large Pfam² familiy PF00472 which currently contains 1549 members, all of which were identified as such PTH domains.³ Many members of this family appear to function in prokaryotic translation termination by hydrolyzing the peptidyl t-RNA ester after polypeptide synthesis is completed. A universally conserved GGQ sequence motif acts as the active site and mimics the aminoacyl stem of peptidyl t-RNA (notably, one of the Glu residues of the motif coordinates the water molecule required for hydrolysis).^4,5

In general, releasing factors are classified based on their specific recognition of tRNA stop codons. Class 1 factors include RF-1 and RF-2 both of which recognize UAA. In contrast, UAG and UGA are specifically recognized only by, respectively, RF-1 and RF-2. Class 2 releasing factors are non codon specific GTP binding proteins and stimulate class 1 releasing factor activity. Five structures of class 1 release factors have thus far been solved: peptide chain RF-1 from E.coli (RF1_ECOLI; PDB ID: 2b3t; sequence identity of 20% with P. syringae PTH domain), peptide chain RF-1 from Thermotoga maritima (RF1_THEMA; PDB ID: 1rq0; sequence identity of 16% with P. syringae PTH domain), immature colon carcinoma transcript 1 protein from Mus Musculus (MOUSE) (ICT1_MOUSE; PDB ID: 1j26, sequence identity of 16% with P. syringae PTH domain), peptide chain RF-1 from Streptococcus mutans (RF1_STRMU; PDB ID: 1zbt, sequence identity of 15% with P. syringae PTH domain) and peptide chain RF-2 from E.coli (RF2_ECOLI; PDB ID: 1gqe; sequence identity of 13% with P. syringae PTH domain). Evidently, the low level of sequence identity across this very large protein domain family prevents the building of good quality homology model for the P. syringae PTH domain. Here we report the NMR solution structure solved by use of a protocol devised for high-throughput NMR protein structure determination.⁶

Materials and Methods

The P. syringae PTH domain was cloned, expressed and purified following standard protocols developed by the NESG for production of uniformly U-¹³C,¹⁵N-labeled protein samples.⁷ First, a domain comprising 130-residues was expressed, but the 2D [¹⁵N,¹H] HSQC spectrum revealed that the C-terminal 30 residues are flexibly disordered in solution. Using these NMR data, the construct was optimized and the 100-residue domain was expressed for NMR structure determination. The corresponding segment of gene PSPTO1818 from P. syringae was cloned into a pET21 (Novagen) derivative, yielding the plasmid Psr211-1-100-21.2. The resulting construct contains eight nonnative residues at the C-terminus (LEHHHHHH) that facilitate protein purification. Escherichia coli BL21 (DE3) pMGK cells, a rare codon enhanced strain, were transformed with PsR211-1-100-21.2, and cultured in MJ9 minimal medium containing (¹⁵NH₄)₂SO₄ and U-¹³C-glucose as sole nitrogen and carbon sources. U-¹³C, ¹⁵N P. syringae PTH domain was purified using an AKTAxpress (GE Healthcare) based two-step protocol consisting of IMAC (HisTrap HP) and gel filtration (HiLoad 26/60 Superdex 75) chromatography. The final yield of purified U-¹³C, ¹⁵N P. syringae PTH domain (> 98% homogenous by SDS-PAGE; 12.7 kDa by MALDI-TOF mass spectrometry) was about 15 mg/L. The final sample of U-¹³C, ¹⁵N labeled P. syringae PTH domain was prepared at a concentration of ~1.2 mM in 95% H₂O/5% D₂O solution containing 5mM CaCl₂, 100 mM NaCl, 20mM NH₄OAc, 10mM DTT, 0.02% NaN₃, 5% D2O, pH 5.5. An isotropic overall rotational correlation time of ~6.5 ns was inferred from ¹⁵N spin relaxation times, indicating that the protein is monomeric in solution. This conclusion was further confirmed by an analytic gel-filtration (Agilent Technologies) followed by a combination of static light scattering and refractive index (Wyatt Technology).⁷

All NMR spectra were recorded at 25 °C on a Varian INOVA 750 spectrometer equipped with a cryogenic probe. Five through-bond correlated G-matrix Fourier transform⁸ (GFT) NMR experiments^8–10 were collected for backbone and side chain resonance assignment (total measurement time: 51 hours), and a 3D ¹⁵N/¹³C^aliphatic/¹³C^aromatic-resolved [¹H,¹H]-NOESY spectrum¹⁰ (mixing time: 70 ms; measurement time: 24 hours) was acquired to derive ¹H-¹H distance constraints. Spectra were processed and analyzed with the programs NMRpipe¹¹ and XEASY¹², respectively. Sequence specific backbone (H^N, H^α, N, C^α) and H^β/C^β resonance assignments were obtained with (4,3)D HNNC^αβC^α/C^αβC^α(CO)NHN⁴ and (4,3)D H^αβC^αβ(CO)NHN experiments using the program AUTOASSIGN.¹³ Side-chain assignments were accomplished by using aliphatic and aromatic (4,3)D HCCH. Overall, assignments were obtained for 99% of routinely assigned backbone (excluding the N-terminal NH₃⁺, the Pro ¹⁵N and the ¹³C’ shifts) and ¹³C^β, and for 98% of the side chain chemical shifts (excluding Lys NH₃⁺, Arg NH₂, OH, side chain ¹³C′ and aromatic quaternary ¹³C shifts; Table I). Stereo-specific assignments were obtained for 38% of the β-methylene groups exhibiting non-degenerate proton chemical shifts using glomsa module of CYANA.¹⁴ 90% of the Val and Leu isopropyl moieties with non-degenerate chemical shifts (Table I) were obtained by using partially ¹³C labeled (5%).¹⁵ Chemical shifts were deposited in the BioMagResBank (accession code: 15471). Upper distance limit constraints for structure calculations were extracted from NOESY (Table I), and backbone dihedral angle constraints were derived from chemical shifts as described¹⁶ for residues located in regular secondary structure elements (Table I) by using the program TALOS. The programs CYANA^14,17 and AUTOSTRUCTURE¹⁸ were used in parallel to automatically assign long-range NOEs. The final structure calculations were performed using version 2.1 of CYANA¹⁷ and CNS¹⁹ with the DYANA constraints (see: http://www.las.jp/prod/cyana/eg).

Table I.

Statistics of NMR Structure of P.syringae PTH domain

Conformationally restricting distance constraints
Intraresidue [I = j]	448
Sequential [(I – j) = 1]	586
Medium Range [1 < (I – j) ≤ 5]	354
Long Range [(I – j) > 5]	541
Total	1929
Dihedral angle constraints
Φ	51
Ψ	51
Number of constraints per residue	20.3
Number of long-range constraints per residue	5.4
Completeness of stereo-specific assignmentsa [%]
βCH2	38 (28/74)
Val and Leu isopropyl groups	90 (19/21)
CYANA target function [Å2]	0.30 ± 0.004
Average r.m.s.d. to the meanCNS coordinates [Å]
regular secondary structure elementsb, backbone heavy	0.58±0.10
regular secondary structure elements, all heavy atoms	0.98±0.10
residues 1–21, 34–102 backbone heavy atoms N, Cα, C′	0.72±0.12
residues 1–21, 34–102 all heavy atoms	1.17±0.12
heavy atoms of molecular core (or best-defined SC)c	0.72±0.11
PROCHECK rawscored (φ and Ψ/all dihedral angles)	−0.09/−0.16
PROCHECK Z-scoresd (φ and Ψ/all dihedral angles)	−0.04/−0.95
MOLPROBITY raw score/Z-scoree	16.64/−1.33
Auto QF R/P/DP scores (%)f	0.97/0.96/0.80
Ramachandran plot summary ordered residue ranges: 7–47, 59–72, 76 – 82, 87 – 91 [%]
most favored regions	93.2
additionally allowed regions	6.7
generously allowed regions	0.1
disallowed regions	0
Average number of distance constraints violations per CYANA conformer [A]
0.2–0.5	0.0
> 0.5	0
Average number of dihedral-angle constraint violations per CYANA conformer [degrees]
> 10	0

^aRelative to pairs with non-degenerate chemical shifts.

^bResidues, 2–4, 8–10, 16–19, 37–42, 69–74 (β strands) 50–58, 80–100 (α-helices).

^cIncludes 32 residues:4, 8, 10, 11, 13, 15, 18–20, 36–38, 43, 45, 46, 48–50, 53, 56, 69, 74, 79, 83, 85, 87, 88, 91,95, 96, 98, 99.

^dScores defined in Ref.²³.

^eScores defined in Ref.²⁴.

^fScores defined in Ref.²⁵.

Results and Discussion

The statistics summarized in Table I indicate that a high-quality NMR structure was obtained for the P. syringae PTH domain (Fig 1; PDB ID: 2jva). The structure exhibits a distinct α/β fold containing five β-strands with residues 2–4, 8–10, 16–19, 37–42, 69–74 (A-E), and two α-helices containing residues 50–58, 80–100 (I-II). β-strands C to E form the central anti-parallel sheet with topology C(↑), D(↓), E(↑). The β-strands A and B form a short β-hairpin structure (residues 1–10), which is in contact with both α-helices and the polypeptide segment connecting α-helix I and β-strand D. α-helix I itself is inserted between β-strands D and E. The large C-terminal α-helix II is attached to the β sheet on the same side of the β-hairpin. The flexible loop of residues 21–35 connects β-strands C and D, and contains the catalytically active GGQ tri-peptide segment (Fig. 1b).

Fig 1.

NMR structure of the P. syringae PTH domain (a): The 20 CYANA conformers with the lowest residual CYANA target function representing the NMR solution structure are shown after superposition for minimal r.m.s.d. of the backbone heavy atoms N, C^α and C′ atoms of regular secondary structure elements (Table 1). (b): Ribbon drawing of the CYANA conformer with the lowest residual target function value (Table I). The α-helices I to II are shown in red and yellow, β strands (A-E) are shown in cyan and other polypeptide segments are in grey, and the N- and C-terminal boundaries of the polypeptide segment are indicated as ‘N’ and ‘C’. Backbone and side chains of universally conserved GGQ (residues 26–28) motif are depicted in blue. (c): ‘Sausage’ representation of 20 superimposed conformers in the orientation of (a). For the presentation of the backbone a spline function was draw through the C^α positions and the thickness of the cylindrical rod is proportional to the mean of the global displacements of the 20 CYANA conformers calculated after superposition as in described in (a). The α-helices are shown in red and other polypeptide segments are displayed in grey. The 32 best-defined side chains (Table I) are also displayed. These figures were generated using the program MOLMOL.²⁶

Fig-2 displays the structures of domains belonging to Pfam family PF00472 that have been solved so far. The sequence identity with the P. syringae PTH domain is 20% or less in all cases. In fact, a search for structurally similar proteins using the programs DALI²⁰ (Z-score >4.0 as a cut-off criterion) and SSM²¹ identified only two of these structures as being structurally similar. Both programs identified the PTH domain of immature colon carcinoma transcript 1 protein from Mus musculus (MOUSE) (Fig. 2D; PDB ID: 1j26, gi:20306640, SwissProt/TrEMBL ID: ICT1_MOUSE, DALI Z-score 7.6, sequence identity of 23% with P. syringae PTH domain, r.m.s.d. of 3.7 A° with residues 6–26, 31–59 and 61–100 of P. syringae PTH domain, SSM Z-score 5.3, sequence identity of 17% with P. syringae PTH domain, r.m.s.d. of 1.89 A° with residues 15–19, 37–42, 49–58, 69–74 and 79–101 of P. syringae PTH domain), which is a thus far functionally uncharacterized protein. The comparison reveals that the P. syringae PTH domain has an additional short β-hairpin. DALI also identified the PTH domain of the polypeptide chain RF-2 from E. coli²² (PDB ID: 1gqe, gi: 146339, SwissProt/TrEMBL ID: RF2_ECOLI, Z-score 4.8, sequence identity of 16% with P. syringae PTH domain, rmsd of 3.9 A° with residues 2–5, 8–25, 30–43, 68–102, 103–108). The comparison reveals that the N-terminal β-hairpin and α-helix I of P. syringae PTH domain (Fig. 2A) are not present in this protein, but the overall similarity reveals that P. syringae PTH domain possess the fold of the PTH domain of class I peptide chain release factor.

Fig 2.

Ribbon diagrams of the six currently known structures belonging to PFam family PF00472. The PTH domains are shown in color and, for structures of full length proteins, the remainder of the protein chain is shown in grey. (A) P. syringae PTH domain (Fig. 1). (B) Immature colon carcinoma transcript 1 protein from Mus Musculus (mouse) (ICT1_MOUSE; PDB ID: 1j26, rmsd: 2.9 A°, sequence identity of 16% with P. syringae PTH domain). (C) Peptide chain RF-2 from E. coli (RF2_ECOLI; PDB ID: 1gqe, rmsd 3.8 A°, sequence identity of 13% with P. syringae PTH domain). (D) Peptide chain RF-1 from E.coli (RF1_ECOLI; PDB ID: 2b3t, rmsd 5.1 A°, sequence identity of 20% with P. syringae PTH domain). (E) Peptide chain RF-1 from Thermotoga maritima (RF1_THEMA; PDB ID: 1rq0, rmsd 4.9 A° sequence identity of 16% with P. syringae PTH domain). (F) Peptide chain RF-1 from Streptococcus mutans (RF1_STRMU; PDB ID: 1zbt, rmsd 4.19 A°, sequence identity of 15% with P. syringae PTH domain).

Taken together, the NMR structure of the P. syringae PTH domain significantly increases the ‘structural coverage’ of the large Pfam family PF00472 and finds 1395 proteins that are sufficiently related in sequence to build very reliable homology models (note that all those proteins belong to the same Pfam family and match at E-values <10–10) of members of this family. Moreover, the comparison of the solution structure of the P. syringae PTH domain with the domain structures in the ribosomal complex suggests that conformational changes upon formation of the complexes are moderate.