Hot spots for allosteric regulation on protein surfaces

Abstract

Recent work indicates a general architecture for proteins in which sparse networks of physically contiguous and coevolving amino acids underlie basic aspects of structure and function. These networks, termed sectors, are spatially organized such that active sites are linked to many surface sites distributed throughout the structure. Using the metabolic enzyme dihydrofolate reductase as a model system, we show that: (1) the sector is strongly correlated to a network of residues undergoing millisecond conformational fluctuations associated with enzyme catalysis, and (2) sector-connected surface sites are statistically preferred locations for the emergence of allosteric control in vivo. Thus, sectors represent an evolutionarily conserved "wiring" mechanism that can enable perturbations at specific surface positions to rapidly initiate conformational control over protein function. These findings suggest that sectors enable the evolution of intermolecular communication and regulation.

Figure 1

The DHFR sector and residues involved in millisecond dynamics relevant to catalysis. A, The reaction cycle of E. coli DHFR. DHFR catalyzes the stereospecific reduction of dihydrofolate (DHF) to tethrahydrofolate (THF) through transfer of a hydride ion from the cofactor NADPH. The main structure change associated with the reaction cycle is a switch between the so-called closed and occluded conformations, a fluctuation that occurs on a similar time scale as the catalytic step of the reaction. B, A mapping of amino acids undergoing conformational exchange at the millisecond time scale in any of the complexes of E. coli DHFR representing the catalytic cycle (shown as small orange spheres on the Cα atom, PDB ID: 1RX2) (Boehr et al., 2006, 2010; McElheny et al., 2005)}. C, The SCA sector for the DHFR family in shown in CPK (blue and orange, and see Table S1). Orange and blue spheres represent sector positions either overlapping or not, respectively, with residues undergoing millisecond dynamics. The small orange spheres represent non-sector positions involved in millisecond dynamics. This analysis shows that sector positions are strongly correlated with residues undergoing dynamical motions underlying catalysis (p < 0.006, see text and Table S2).

Figure 2

Sector architecture in DHFR. A-B, Two views (different by 90° rotation) of the surface (A) and a slice through the protein core (B) with sector residues colored in blue. Substrate and cofactor are shown in yellow and green stick bonds, respectively. C, A cartoon representation of the slice mappings in B, illustrating that the sector comprises a sparse, physically connected network of residues that link the active site to a few distant surface positions (red).

Figure 3

Comprehensive domain insertion scan and relative growth rate measurements by high-throughput sequencing. A, All LOV2 domain insertion sites on the DHFR surface, (70 in total, orange spheres). For simplicity in discussion, we refer to each DHFR-LOV2 chimera or DHFR mutant as a “variant”. B, Barcoding strategy for the DHFR variants. Each DHFR mutant or DHFR-LOV2 chimera was labeled with two DNA barcodes: (1) a five-base pair barcode that identifies the time point of sampling and experimental condition (dark or lit), and (2) a five base pair barcode immediately following the DHFR stop codon that identifies the variant. The first barcode was added to the 5′ end of the sequenced region during sample preparation by PCR (see methods). Sequencing of both barcodes permits determination of relative variant frequencies within a mixed population as they vary with time and experimental condition. C, Measurement of growth rates through sequencing for a set of DHFR point mutants that span a broad range of catalytic activities in vitro. The log frequency of each variant is shown relative to wild type, and is normalized to the initial values at the start of the experiment (t=0, see Methods). Thus, slopes of the linear regression report growth rates relative to wild-type. D, Comparison between in vitro catalytic power and in vivo relative growth rate, indicating a monotonic relationship between the two (see Table S3 and Figures S1 and S2). Yellow and black circles represent two independent experimental trials in the light and dark respectively.

Figure 4

Light-dependence in growth rate for one chimera, DL121, previously shown to display weak light-dependence in catalytic rate in vitro. A-B, Experiments under dark and lit conditions show that DL121 displays an ~16% increase in growth rate in response to light, while two DL121 variants carrying LOV2 domains defective in allosteric mechanism (121-C450S and 121-noJ) do not show light dependence (Fig. S3). C, Quantitative measurement of light-dependence in growth rate in three independent growth/sequencing experiments for 10 non light-dependent DHFR mutants spanning a broad range of catalytic power, and for DL121, 121-C450S, and 121-noJ. Error bars indicate the standard error of the mean across the three experiments. The data demonstrate good reproducibility in growth rate measurements in independent experiments, and establish a statistical model for measurement noise in this assay based on the behavior of the non light-dependent mutants of DHFR (the dashed lines indicate 2σ deviation from the mean). In comparison, DL121 shows clear statistically significant light-dependence, while 121-C450S and 121-noJ do not.

Figure 5

The emergence of allosteric control at sector-connected DHFR surface sites. A, Histograms of growth rate difference (lit-dark) for all DHFR-LOV2 chimeras (black) and non light-dependent DHFR mutants (grey, with Gaussian fit in red). B-C, growth rate differences for non light-dependent mutants (B) and for DHFR-LOV2 chimeras ordered by DHFR primary structure (C) (see also Fig. S4). Error bars indicate standard error of the mean across three experimental repeats. The corresponding secondary structure pattern is indicated at right. The grey/red bars at right indicate the position of each LOV2 insertion; red bars indicate insertions sites showing light-dependence (Z > 2) of non light-dependent controls (Table S4). Light dependent positions are scattered throughout the primary and secondary structure of the protein. D, Correlation of light dependence in vitro and in vivo. The catalytic rate (k_cat) was measured for four DHFR-LOV2 fusions under lit and dark conditions. This confirms that DL121, DL127, and DL134 show light dependence in enzymatic activity as measured biochemically. E, Mapped on an atomic structure of E.coli DHFR, light -dependent positions (red) comprise a spatially distributed subset of the protein surface positions (light blue). F, Statistics of DHFR positions showing sector connectivity and light-dependence. Every light-dependent position is also sector connected over a range of significance thresholds for light-dependence and sector definition (Table S5). These results indicate robust statistical correlation between sector connectivity and capacity for allosteric control.

Figure 6

Pathways of sector connectivity between the active site and light-dependent surface positions. A, Space filling representation of DHFR with light dependent surface positions in red. B-F, serial slices taken through DHFR at the planes indicated in panel A; the views in B-F are from the left. The data show that sectors form physically contiguous pathways through the core of the three-dimensional structure that connect all light-dependent positions with the substrate and cofactor binding sites and with the catalytic active site. Substrate and cofactor are shown as yellow or green stick bonds, respectively. Thus, light-dependent positions are “wired up” to the active site through sector amino acids.

Figure 7

Sector connected surfaces modulate function in the PDZ domain. A, Space filling representation of PDZ with surface mutations that perturb protein function in red. B-F, serial slices taken through PDZ at the planes indicated in panel A; the views in B-F are from the left. As for DHFR, the data show that sectors form physically contiguous pathways through the structure that connect all mutations that impact PDZ function to the peptide binding site. The peptide is indicated in yellow stick bonds.