NMR Structure of Protein YvyC from Bacillus subtilis Reveals Unexpected Structural Similarity between Two PFAM Families

Introduction

109-residue protein YvyC (MW=13 kDa) from B. subtilis (gi|580862, SwissProt/TrEMBL ID YVYC_BACSU, accession number P39737) was selected as a target of the Protein Structure Initiative 2 and assigned to the Northeast Structural Genomics consortium (NESG; http://www.nesg.org) for structure determination (NESG Target ID SR482). YvyC belongs to the Pfam¹ protein family FlaG (PF03646) and is required for proper assembly of bacterial flagella. The FlaG family contains 215 members (for sequence alignment of the FlaG family seeds see Fig. S1 in the Supporting Information). The NMR structure of YvyC presented here is the first atomic resolution structure available for this family.

Self-assembly of bacterial flagella has been studied extensively in recent decades and comprehensive reviews are available.^2-4 The gene encoding YvyC is a part of the fliD operon comprising the genes yvyC, fliD, fliS and fliT.⁵ This operon is located immediately downstream of the gene hag (also called fliC) encoding flagellin – the major protein required for assembly of the flagellar filaments. During filament elongation, flagellin monomers are exported through the central channel of a flagellum and oligomerize at its distal end.^2,4 FliD (also named ‘hook-associated protein 2’, or HAP2⁶) forms a pentameric cap at the distal end of a flagellum and is essential for oligomerization of flagellin.^2,4,7 FliS and FliT serve as flagellin-specific⁸ and FliD-specific^9,10 chaperones, respectively. It was demonstrated that mutations in yvyC gene orthologs in P. fluorescens (flaG) and V. anguillarum (ORF3) lead to phenotypes with unusually long filaments,^11,12 but the specific role of YvyC remains unknown.

Materials and Methods

YvyC was cloned, expressed and purified following standard automated protocols to produce a uniformly ¹³C,¹⁵N-labeled protein sample.^13,14 Briefly, the full length yvyC gene from Bacillus subtlis was cloned into a pET21d (Novagen) derivative, yielding the plasmid pSR482-21.1. The resulting construct contains eight nonnative residues at the C-terminus (LEHHHHHH) to facilitate protein purification and one residue insertion (L) following the initiation codon introduced by cloning. Escherichia Coli BL21 (DE3) pMGK cells, a rare codon enhanced strain, were transformed with pSR482-21.1, and cultured in MJ9 minimal medium containing (¹⁵NH₄)₂SO₄ and U-¹³C-glucose as sole nitrogen and carbon sources. U-¹³C,¹⁵N YvyC was purified using an AKTA Express (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 YvyC (> 97% homogeneous by SDS-PAGE; 15.0 kDa by MALDI-TOF mass spectrometry) was ∼48 mg/L. In addition, a U-¹⁵N and 5% biosynthetically directed fractionally ¹³C-labeled sample¹⁵ was generated for stereo-specific assignment of isopropyl methyl groups. U-¹³C,¹⁵N and 5%¹³C,U-¹⁵N YvyC were dissolved, respectively, at concentrations of ∼1.0 mM and 1.1 mM in 95% H₂O/5% D₂O (20 mM MES, 100 mM NaCl, 10 mM DTT, 5 mM CaCl₂, 0.02% NaN₃) at pH 6.5. An isotropic overall rotational correlation time of ∼8 ns was inferred from ¹⁵N spin relaxation times, indicating that the protein is monomeric in solution under the conditions used for these NMR studies. This conclusion was further confirmed by analytic gel-filtration in 100 mM Tris, 100 mM NaCl, 250 ppm NaN₃, at pH 7.5, with detection using a combination of static light scattering and refractive index (as described in ref 12); under these conditions the sample was observed to be > 97% monomeric.

All NMR spectra were recorded at 25 °C. Five G-matrix Fourier transform (GFT) NMR experiments¹⁷ and a simultaneous 3D ¹⁵N/¹³C^aliphatic/¹³C^aromatic-resolved NOESY¹⁸ spectrum (mixing time 60 ms) were acquired on a Varian INOVA 750 MHz spectrometer equipped with a conventional probe. 2D constant-time [¹³C, ¹H]-HSQC spectra with 28 ms and 56 ms constant-time delays were recorded for the 5% biosynthetically directed fractionally ¹³C-labeled sample on a Varian INOVA 600 MHz spectrometer equipped with a cryogenic probe in order to obtain stereo-specific assignments for isopropyl groups of valines and leucines.¹⁵ Spectra were processed using the program PROSA¹⁹ and analyzed using the program CARA.²⁰ Sequence-specific backbone (H^N, H^α, N, C^α) and H^β/C^β resonance assignments were obtained by using (4,3)D HNNC^αβC^α / C^αβC^α(CO)NHN and (4,3)D H^αβC^αβ(CO)NHN along with the program AutoAssign program.²¹ Side-chain spin system identification was accomplished by using aliphatic^16,17 and aromatic²² (4,3)D HCCH. Assignments were obtained for 100% of backbone and side-chain chemical shifts assignable with the NMR experiments listed above (excluding N-terminal NH₃⁺, Lys NH₃⁺, Arg NH₂, OH of Ser, Thr and Tyr, ¹³C^ε of Asp and Asn, ¹³C^δ of Glu and Gln, and aromatic ¹³C^γ shifts; Table I). Stereo-specific assignments were obtained for all Val and Leu methyl groups and for 40% of the β-methylene groups exhibiting non-degenerate chemical shifts (Table I). Chemical shifts were deposited in the BioMagResBank on 06/15/2006 with accession code 7170.

TABLE I. Statistics of NMR Structure of Protein YvyC.

Completeness of stereo-specific assignmentsa [%]
αCH2 of Gly	100 (1/1)
βCH2	40 (28/70)
Val and Leu methyl groups	100 (22/22)
Conformationally restricting distance constraints
Intraresidue [i = j]	548
Sequential [|i – j| = 1]	728
Medium Range [1 < |i – j| < 5]	697
Long Range [|i – j| ≥ 5]	916
Total	2889
Dihedral angle constraints
φ	35
ψ	35
Average number of constraints per residue	25.8
Average number of long-range distance constraints per residue	8.2
CYANA target function [Å2]	2.33±0.12
Average number of distance constraints violations per CYANA conformer
0.2 – 0.5 Å	0
> 0.5 Å	0
Average number of dihedral-angle constraint violations per CYANA conformer
> 5°	0
Average r.m.s.d. to the mean CYANA coordinates [Å]
Regular secondary structure elementsb, backbone heavy atoms	0.60±0.10
Regular secondary structure elementsb, all heavy atoms	1.00±0.11
Ordered residuesc, backbone heavy atoms	0.97±0.36
Ordered residuesc, all heavy atoms	1.43±0.35
Heavy atoms of molecular core including best-defined side chainsd	0.70±0.09
PROCHECK37 G-factors raw score (φ and ψ / all dihedral angles) c	-0.15/-0.20
PROCHECK37 G-factors Z-score (φ and ψ / all dihedral angles) c	-0.28/-1.18
MOLPROBITY38 clash score (raw / Z-score)c	24.03/-2.66
AutoQF R/P/DP scores39 [%]	95/95/85
Ramachandran plot summary (residues 2-17, 37-104) c [%]
most favored regions	91.2
Additionally allowed regions	7.8
generously allowed regions	0.4
disallowed regions	0.5

^aRelative to pairs with non-degenerate chemical shifts.

^bResidues 10-19, 37-49, 58-65, 68-75, 80-85, 90-101.

^cResidues 3-5, 8-21, 38-55, 58-64, 67-104, 108-111. Ordered residues were defined based on dihedral angle order parameters, and Z-scores were computed relative to corresponding structure quality measures for high resolution X-ray crystal structures, as described in reference.⁴⁰

^dBackbone and side-chain heavy atoms of residues 3, 6, 10-13, 16, 41, 43-45, 47, 48, 50, 51, 53, 54, 59, 62, 63, 71-74, 78, 81, 82, 85-87, 90, 94-97, 102, 103.

¹H–¹H upper distance limit constraints for structure calculations were obtained from NOESY (Table I). In addition, backbone dihedral angle constraints were derived from chemical shifts using the program TALOS²³ for residues located in well-defined secondary structure elements (Table I). The programs CYANA^24,25 and AUTOSTRUCTURE²⁶ were used in parallel to assign NOEs by consensus, and the remaining assignments were carrieMOVEd by interactive spectral analysis.²⁷ The final structure calculation was performed with CYANA, and the 20 conformers with the lowest target function value were refined in an ‘explicit water bath’²⁸ using the program CNS.²⁹ The coordinates were deposited in the Protein Data Bank on 06/15/2006 (accession code 2HC5).

Results and Discussion

A high-quality NMR structure of protein YvyC (Table I) was obtained. The structure consists of three α-helices I-III (residues 9-22, 36-52 and 86-105) and three β-strands (residues 58-65, 68-75 and 81-85) forming one anti-parallel β-sheet with topology A(↑), B(↓), C(↑) (Fig. 1b). The helices form a three-helix bundle, which is attached to one side of the sheet. The secondary structure elements are locally and globally well-defined. The segment comprising residues 23-35 connecting helices I and II is largely disordered (Fig 1a,c), which is manifested by comparably narrow NMR lines and lack of medium- and long-range NOEs.

Fig. 1.

NMR structure of YvyC. (a) Backbone trace of residues 1-104 of the 20 representative CYANA conformers after superposition of backbone N, C^α and C′ atoms of the regular secondary structure elements for minimal root-mean-square deviation (RMSD). (b) Ribbon drawing of residues 1-104 of the conformer with the lowest CYANA target function. α-helices I-III are shown in red and yellow, β-strands A-C are shown in cyan, other polypeptide segments are shown in gray and the N- and C-termini are labeled as “N” and “C”. (c) Sausage representation of backbone and best defined side chains. A spline curve was drawn through the mean positions of C^α atoms of residues 1-104 with the thickness proportional to the mean global displacement of C^α atoms in the 20 conformers superimposed in (a). α-helices I-III are shown in red, β-strands A-C are shown in cyan, other polypeptide segments are shown in gray and a superposition of 34 side chains with the lowest global displacement is shown in blue. (d) Ribbon drawing of the conformers with the lowest CYANA target function of YvyC (pale green) and YkfF (grey) after superposition of Ca atoms of residues 37-46, 60-63, 70-74, 81-85, 90-98 and 14-23, 27-30, 38-42, 48-52, 59-67, respectively. For YvyC only residues 1-104 are shown. (e) Surface representation of the conformer with the lowest CYANA target function. The structure shown on the left has the same orientation as in (a-c), while the one on the right is rotated by 180° about the vertical axis. Surface colors represent sequence conservation among the seed sequences of the FlaG protein family calculated with ConSurf,³³ with burgundy corresponding to the highest conservation and cyan – to the highest variability. The cavities are labeled as C1, C2 and C3. (f) Same as (e), but with the colors according to the electrostatic potential. All figures were prepared with the program MOLMOL.³⁵

A search of the CATH database using the CATHEDRAL³⁰ server did not yield a matching fold for protein YvyC, indicating that YvyC exhibits a distinct protein architecture. Moreover, a search of the PDB database for structurally similar proteins using both DALI³¹ and SSM³² identified protein YkfF from E. coli (NESG target ER397, PDB code 2HJJ) as the only significant (DALI Z-score = 3.3) hit. The structurally aligned fragment comprises 56 residues (r.m.s.d. of C^α atoms = 2.9 Å; sequence identity 5%) and contains five regular secondary structure elements (the β-sheet with strands A-C and α-helices II and III, Fig 1d). YkfF is the sole structural representative of Pfam family PF06006 comprising 38 proteins of unknown function. Hence, structure comparison of proteins YvyC from B. Subtilis and YkfF from E. coli indicates a possible distant homology between the thus far unrelated PFAM families PF03646 and PF06006.

Analysis of conserved surface features³³ within the FlaG protein family PF03646 reveals a single cluster of highly conserved residues on the surface of YvyC (Fig. 1e). These residues belong to β-strands B and C. Furthermore, a search for protein surface cavities using the ProFunc³⁴ server revealed three clefts (Fig. 1e,f) with volumes of ∼1,800 ų, ∼800 ų and ∼660 ų. The calculation of the electrostatic protein surface potential calculated with the program MolMol³⁵ shows that in protein YvyC cavity 2 is charged negatively while cavities 1 and 3 exhibit a mixed charge distribution (Fig. 1f). Considering (i) that residues Leu 58, Glu 75, Ile 82 in cavity 1 and Glu 83 and Pro 86 in cavity 3 are highly conserved and (ii) that functional sites on protein surfaces are mostly located in the largest cavities³⁶, one may conclude that these two cavities are likely to be important for the molecular function.