Solution NMR structure of Alr2454 from Nostoc sp. PCC 7120, the first structural representative of Pfam domain family PF11267

Abstract

Protein domain family PF11267 (DUF3067) is a family of proteins of unknown function found in both bacteria and eukaryotes. Here we present the solution NMR structure of the 102-residue Alr2454 protein from Nostoc sp. PCC 7120, which constitutes the first structural representative from this conserved protein domain family. The structure of Nostoc sp. Alr2454 adopts a novel protein fold.

Introduction

We present the solution NMR structure of the 102-residue Alr2454 protein from Nostoc sp. strain PCC 7120 (UniProtKB/TrEMBL ID, Q8YUA0_NOSS1; NESG ID, NsR264), a member of the functionally uncharacterized Pfam protein domain family PF11267 (DUF3067) comprised of bacterial and eukaryotic sequences, predominantly from cyanobacteria and viridiplantae (green plants), respectively (Fig. 1A) [1, 2]. This protein was selected for three-dimensional structure determination by the Northeast Structural Genomics Consortium (NESG) as part of the second phase of the Protein Structure Initiative (PSI-2) aimed at providing structural coverage of large, uncharacterized protein domain families [3]. Initial structural representatives of such families exhibit high homology modeling leverage [4], provide insights into protein evolution, and expand our knowledge of fundamental relationships between protein sequences, 3-dimensional structure, and protein function. The structure of Nostoc sp. Alr2454, the first structural representative from the PF11267 protein domain family, features a unique and, to the best of our knowledge, novel protein fold.

Fig. 1.

Solution NMR structure of Alr2454 from Nostoc sp. PCC 7120 (PDB ID, 2LJW). (A) Sequence alignment of a representative subset of the Pfam PF11267 protein domain family [1] from cyanaobacteria (Nostoc sp., Q8YUA0_NOSS1; Trichodesmium erythraeum, Q114L5_TRIEI; Synechococcus elongatus, Q31PB2_SYNE7; Prochlorococcus marinus, A2BVC5_PROM5, residues 4 to 105) and plants (Arabidopsis thaliana, Q8LDI1_ARATH, residues 113 to 222; Zea mays, B4FUD0_MAIZE, residues 116 to 225). Sequences were aligned using Clustal W 2.0 [33] and the sequence alignment was rendered using ESPript [34]. Boxed residues represent identical (white font, highlighted in red) and similar (red font) amino acid conservation. Residue numbering for Nostoc sp. Alr2454 and secondary structural elements [26] from its solution NMR structure (PDB ID, 2LJW) are drawn above the alignment. (B) Superposition of the final ensemble of 20 conformers of Alr2454 (residues 1 to 102). The view of the β-face of the protein is shown; α-helices and β-strands are shown in cyan and magenta, respectively, and loops are colored grey. (C) Stereoview of the lowest energy (CNS) conformer of Alr2454. Same orientation and color scheme as in (B). Secondary structural elements are labeled. (D) ConSurf [27, 28] image showing the conserved residues in Alr2454 (residues 3 to 101). Residue coloring, reflecting the degree of residue conservation over the entire PF11267 protein domain family (Pfam 25.0 [1]; 80 sequences), ranges from magenta (highly conserved) to cyan (variable). (Left) β-sheet face of the protein. (Right) α-helical face of the protein. Selected highly conserved residues are labeled. (E) APBS [29, 30] solvent accessible electrostatic surface potential of the β-sheet face of Alr2454 (residues 1 to 102) showing negative (red), neutral (white), and positive (blue) charges. All structure figures were rendered using PyMOL (www.pymol.org).

Material and Methods

Protein purification

Isotopically enriched samples of Nostoc sp. Alr2454 ([U-¹³C,¹⁵N]- and [U-5%-¹³C,100%-¹⁵N]-Alr2454) for NMR spectroscopy were cloned, expressed, and purified following standard NESG protocols [5]. Briefly, the 102-residue coding sequence of the alr2454 gene from Nostoc sp. strain PCC 7120 was cloned into the pET21_NESG vector (Novagen) in frame with a C-terminal affinity tag (LEHHHHHH), transformed into codon-enhanced Escherichia coli BL21 (DE3) pMGK cells, and expressed in MJ9 minimal medium [6] containing U-(¹⁵NH₄)₂SO₄ and U-¹³C-glucose as the sole nitrogen and carbon sources. Initial cell growth was carried out at 37 °C, and protein expression was induced at 17 °C by 1 mM IPTG at mid-log phase growth and continued overnight. Proteins were purified using an ÄKTAxpress system (GE Healthcare) with a two-step protocol consisting of HisTrap HP affinity chromatography followed by HiLoad 26/60 Superdex 75 gel filtration chromatography. The final yield of purified isotopically-enriched Alr2454 was approximately 40 mg/L of culture. Sample purity and molecular mass were confirmed by SDS-PAGE and MALDI-TOF mass spectrometry (MALDI-TOF mass of [U-¹³C,¹⁵N]-Alr2454 (Da): experimental, 13,733; expected, 13,724). Samples of [U-¹³C,¹⁵N]- and [U-5%-¹³C,100%-¹⁵N]-Alr2454 for NMR spectroscopy were concentrated by ultracentrifugation to 0.6 to 1.0 mM in 90% H₂O/10% 2H₂O solution containing 20 mM ammonium acetate, 100 mM NaCl, 10 mM DTT, 5 mM CaCl₂, 50 μM DSS at pH 4.5. Analytical gel filtration followed by static light scattering (Suppl. Fig. S1) and ¹⁵N T₁ and T₂ relaxation data (Suppl. Fig. S2) demonstrate that Alr2454 is a monomer in solution under the conditions used in the NMR studies. The pET expression vector for Nostoc sp. Alr2454, (NESG NsR264-21.4), has been deposited in the PSI Materials Repository (http://psimr.asu.edu/).

NMR spectroscopy and resonance assignment

All NMR data were collected at 298 K on Bruker AVANCE 600 and 800 MHz spectrometers equipped with 1.7-mm TCI and 5-mm TXI cryoprobes, respectively, processed with NMRPipe [7], and visualized using SPARKY [8]. All spectra were referenced to internal DSS. Complete ¹H, ¹³C, and ¹⁵N resonance assignments for Alr2454 were determined using conventional triple resonance NMR methods. Backbone resonance assignments were analyzed automatically using both PINE 1.0 [9] and AutoAssign 2.4.0 [10] software, based on peak lists for 2D ¹H-¹⁵N HSQC and 3D HNCO, HN(CA)CO, HN(CO)CA, HNCA, CBCA(CO)NH and HNCACB spectra. The assigned ¹H-¹⁵N HSQC spectrum of Alr2454 is provided as Suppl. Fig. S3, and a summary of the sequential connectivity and NOESY data used to determine these assignments as Suppl. Fig. S4. Side chain assignments were completed manually using 3D HBHA(CO)NH, HCCH-COSY, HCCH-TOCSY, and (H)CCH-TOCSY experiments. Stereospecific isopropyl methyl resonance assignments for all Val and Leu residues were determined from characteristic cross-peak fine structures in high resolution 2D ¹H-¹³C HSQC spectra of [U-5%-¹³C,100%-¹⁵N]-Alr2454 [11]. Resonance assignments were validated using the Assignment Validation Suite (AVS) software package [12]. The final resonance assignments, NOESY spectral peak lists, and time domain data for Alr2454 were deposited in the BioMagResDB (BMRB accession number, 17965). ¹H-¹⁵N heteronuclear NOE and ¹⁵N T₁ and T₂ relaxation measurements were made using gradient sensitivity-enhanced 2D heteronuclear NOE and 1D ¹⁵N T₁ and T₂ (CPMG) relaxation experiments, respectively [13].

Structure determination and refinement

The solution NMR structure of Alr2454 was calculated using CYANA 3.0 [14, 15] supplied with peak intensities from 3D ¹⁵N-edited NOESY (τ_m = 100 ms), 3D 13C-edited aliphatic NOESY (τ_m = 100 ms), and 3D ¹³C-edited aromatic NOESY (τ_m = 120 ms) spectra, together with broad dihedral angle constraints derived by TALOS+ [16] (φ, ψ ± 30°) for ordered residues with confidence scores of 10. The 20 structures with lowest target function out of 100 calculated in the final cycle were further refined by restrained molecular dynamics in explicit water using CNS 1.3 [17, 18] and the PARAM19 force field, supplied with the final NOE-derived distance and TALOS+ dihedral angle constraints. In this final stage of the structure determination, rotamer states of specific ordered residues were constrained (χ₁, χ₂ ± 20°) based on MolProbity [19, 20] and PROCHECK [21] analyses.

Structure validation and deposition

The final refined ensemble of 20 structures for Alr2454 (excluding the C-terminal His₆ tag) was deposited into the Protein Data Bank (PDB ID, 2LJW). Structural statistics and global structure quality scores, including Verify3D [22], ProsaII [23], PROCHECK [21], and MolProbity [19, 20] raw and statistical Z-scores, were computed using the PSVS 1.4 software package [24]. The global goodness-of-fit of the final structure ensemble with the NOESY peak list data and resonance assignments was determined using the RPF analysis program [25].

Results and Discussion

The solution NMR structure of Nostoc sp. PCC 7120 Alr2454 features a unique mixed α+β fold comprised of four α-helices (α1, Gly3-Trp14; α2, Glu47-Leu64; α3, Ala67-Gln76; α4, Gly94-Ile101) and a sheet of three anti-parallel β-strands (β1, Tyr18-Thr25; β2, Lys28-Tyr37; β3, Val87-Leu91) arranged in a αββααβα topology (Fig. 1B, C) [26]. The three-stranded β-sheet packs against the first three helices (α1 to α3) to form a compact structure, whereas the C-terminal α-helix (α4) is somewhat poorly-defined. Structural statistics for Alr2454 are presented in Table 1.

Table 1.

Summary of structural statistics^a

		Alr2454
Completeness of resonance assignments b:
Backbone (%)		99.4
Side chain (%)		98.3
Aromatic (%)		96.6
Stereospecific methyl (%)		100
Conformationally-restricting constraintsc:
Distance constraints
Total		2478
intra-residue (i = j)		688
sequential (|i−j| = 1)		619
medium range (1 < |i − j| < 5)		462
long range (|i − j| ≥ 5)		709
Dihedral angle constraints		162
Hydrogen bond constraints		0
No. of constraints per residue		25.4
No. of long range constraints per residue		6.8
Residual constraint violationsc:
Average no. of distance violations per structure:
0.1 – 0.2 Å		8.75
0.2 – 0.5 Å		1.85
> 0.5 Å		0
Average no. of dihedral angle violations per structure:
1 – 10°		8.75
> 10°		0
Model Qualityc:
RMSD backbone atoms (Å)d		0.6
RMSD heavy atoms (Å)d		0.9
RMSD bond lengths (Å)		0.018
RMSD bond angles (°)		1.1
MolProbity Ramachandran statisticsc,d
most favored regions (%)		96.8
allowed regions (%)		3.1
disallowed regions (%)		0.1
Global quality scores (Raw/Z-score)c
Verify3D	0.40	−0.96
ProsaII	0.66	0.04
Procheck (phi-psi)d	−0.15	−0.28
Procheck (all)d	−0.03	−0.18
MolProbity clash score	12.51	−0.62
RPF Scorese
Recall/Precision	0.976	0.934
F-measure/DP-score	0.955	0.817
Model Contents:
Ordered residue ranged	1–100
BMRB accession number:	17965
PDB ID:	2LJW

^aStructural statistics computed for the ensemble of 20 deposited structures.

^bComputed using AVS software [12] from the expected number of resonances, excluding: highly exchangeable protons (N-terminal, Lys, and Arg amino groups, hydroxyls of Ser, Thr, Tyr), carboxyls of Asp and Glu, non-protonated aromatic carbons, and the C-terminal His₆ tag.

^cCalculated using PSVS 1.4 [24]. Average distance violations were calculated using the sum over r⁻⁶.

^dBased on ordered residue ranges [S(phi) + S(psi) > 1.8].

^eRPF scores [25] reflecting the goodness-of-fit of the final ensemble of structures (including disordered residues) to the NOESY data and resonance assignments.

ConSurf [27, 28] and electrostatic surface potential [29, 30] analyses of the Alr2454 structure reveal that amino acid residues conserved across the PF11267 protein domain family are primarily clustered on the somewhat basic βsheet face of the protein, particularly in the loops between secondary structural elements (Fig. 1D, E). On the basis of Skan [31] and Dali [32] structural alignment analyses, the structure of Nostoc sp. Alr2454 shows no significant similarity to any protein structure reported to date (Dali Z-scores < 3). Hence, we conclude that Alr2454 adopts a unique protein fold. Moreover, the structure of Alr2454 is sufficiently remote from other known protein structures to preclude a useful prediction of the function of this protein domain family.

An important goal in the PSI program has been to provide novel modeling leverage [4]; i.e., the number of proteins for which homology models can be made using a subject protein structure as a template, that could not be made given the structures in the PDB on the date the subject structure was deposited. Based on criteria for homology modeling described by Liu et al. [4], the Alr2454 structure reported here has a novel homology modeling leverage of 95 structures, including all members of Pfam domain family PF11267.

In summary, the solution NMR structure of Nostoc sp. Alr2454 reported here provides the first structural representative from the Pfam PF11267 (DUF3067) protein domain family of unknown function. In addition, the structure represents a novel protein fold. However, uncovering the exact function of the PF11267 protein domain family awaits further study.

Abbreviations

DSS

2,2-Dimethyl-2-silapentane-5-sulfonic acid

DTT

Dithiothreitol

HSQC

Heteronuclear single quantum coherence

IPTG

Isopropyl β-D-1-thiogalactopyranoside

NOE

Nuclear Overhauser effect

NESG

Northeast Structural Genomics Consortium

PDB

Protein Data Bank

RMSD

Root-mean-square-deviation