Automated protein fold determination using a minimal NMR constraint strategy

Abstract

Determination of precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium-accuracy fold information can often provide key clues about protein evolution and biochemical function(s). In this article we describe a largely automatic strategy for rapid determination of medium-accuracy protein backbone structures. Our strategy derives from ideas originally introduced by other groups for determining medium-accuracy NMR structures of large proteins using deuterated, (13)C-, (15)N-enriched protein samples with selective protonation of side-chain methyl groups ((13)CH(3)). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side-chain (15)N, H(N) resonances, and side-chain (13)CH(3) methyl resonances. These assignments are determined automatically by the program AutoAssign using backbone triple resonance NMR data, together with Spin System Type Assignment Constraints (STACs) derived from side-chain triple-resonance experiments. The program AutoStructure then derives conformational constraints using these chemical shifts, amide (1)H/(2)H exchange, nuclear Overhauser effect spectroscopy (NOESY), and residual dipolar coupling data. The total time required for collecting such NMR data can potentially be as short as a few days. Here we demonstrate an integrated set of NMR software which can process these NMR spectra, carry out resonance assignments, interpret NOESY data, and generate medium-accuracy structures within a few days. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A.

Figure 1.

¹³C-¹H HSQC spectrum of Z domain (at 600 MHz). The ¹³C-¹H HSQC spectrum (red) of ¹³C, ¹⁵N, ²H-enriched, ¹³C-¹H methyl protonated Z domain used in this study is overlaid onto the ¹³C-¹H HSQC spectrum (black) of a fully protonated ¹³C,¹⁵N-enriched sample. The isopropyl methyl groups of all Val, Leu, and δ methyl groups of all Ile (top right) are highly protonated, while the rest of the aliphatic and aromatic carbons are primarily deuterated.

Figure 2.

Sequential connectivity map summarizing the results of automated backbone resonance assignments determined by AutoAssign. Intra (i, red) and sequential (s, yellow) connectivity data used by AutoAssign to establish resonance assignments at each sequence position are shown. Secondary structure information derived automatically from combined analysis of C^α, C^β, and C′ chemical shift (CSI) and NOE data is also plotted along the protein sequence. Shown in the figure are also i to i + 1, i + 2, and i + 3 H^N-H^N NOE connections determined by an initial AutoStructure analysis (run with RDC data) that was used to further validate these assignments; the strength (intensity) of the corresponding NOE interactions are indicated by line thickness. Residues with intra (down triangles) STACi and sequential (up triangles) STACs constraints (derived from hCCcoNH-TOCSY and HcccoNH-TOCSY spectra) specifying N–H^N roots belonging to or following Val, Leu, Ile residues, respectively, are also indicated.

Figure 3.

Ribbon representation and backbone trace of Z domain structures. (A) High- resolution solution NMR structure of Z domain (Tashiro et al. 1997; PDB ID 2SPZ). Structures generated automatically with the “minimal-constraint” strategy outlined in the text (B) without RDC and (C) with RDC data, superimposed on backbone structure of 2SPZ (gold).

Figure 4.

Structures determined by simulated or archived, edited experimental minimal NMR constraints. Coordinate sets for six proteins were taken from Protein Data Bank (PDB). Distance constraints were generated for all interatomic distances <4.5 Å involving amide (backbone N and side-chain N of Asn/Gln) and methyl C of Leu, Val, and Ile(δ). Dihedral angle restraints (for regular secondary structures only), backbone hydrogen bonds, and both N–H^N and C^α–C′ RDC data were also back calculated from the atomic coordinates, as described in the text. These data sets are referred to as Simulated Constraint Data. A second set of constraint lists referred as Archived, Edited Constraint Data were generated by editing archived NMR constraint files deposited in PDB, to replace simulated distance constraints with NOE-derived distances between amide and methyl protons. Other simulated data were also replaced with experimental data where available, as described in the text. For each protein used in this analysis, the published high-resolution NMR structure (PDB structure) is shown along with representative structures from the ensembles generated with CNS using the Simulated Constraint Data or the Archived, Edited Constraint Data.