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Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Schumann FH, Varadan R, Tayakuniyil PP, Grossman JH, Camarero JA, Fushman D - Front Chem (2015)

Bottom Line: On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site.In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work.These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland College Park, MD, USA.

ABSTRACT
Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

No MeSH data available.


Related in: MedlinePlus

Comparison of the 3D structure of the backbone for the circular SH3 constructs with the solution and the (ligand-bound) crystal (Wu et al., 1995) structures for the linear domain. The solution structures are represented by the mean structure for each ensemble, colored green (SH3lin-wt), cyan (SH3circ-Δ), blue (SH3circ-GΔ), and yellow (SH3circ-wt). The crystal structure is colored red. These structures were superimposed using backbone heavy atoms for the core residues. The two panels represent two different orientations of the protein: (A) similar to that in Figure 1 and (B) view from the bottom. Elements or secondary structure and the termini are indicated. The drawings were made using InsightII.
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Figure 7: Comparison of the 3D structure of the backbone for the circular SH3 constructs with the solution and the (ligand-bound) crystal (Wu et al., 1995) structures for the linear domain. The solution structures are represented by the mean structure for each ensemble, colored green (SH3lin-wt), cyan (SH3circ-Δ), blue (SH3circ-GΔ), and yellow (SH3circ-wt). The crystal structure is colored red. These structures were superimposed using backbone heavy atoms for the core residues. The two panels represent two different orientations of the protein: (A) similar to that in Figure 1 and (B) view from the bottom. Elements or secondary structure and the termini are indicated. The drawings were made using InsightII.

Mentions: Our chemical shift data and structure calculations both indicate that structural perturbations in the SH3 domain caused by circularization are small. Figure 7 presents a superposition of the three-dimensional structures of all SH3 constructs studied here and of the crystal structure of linear SH3 in the bound form. The backbone fold is very similar in all these SH3 constructs; the pair-wise RMSDs between the mean NMR structures (for each NMR ensemble) are 0.8–1.1 Å and reduce to 0.3–0.6 Å if only core residues are considered (Table 3). As expected, the termini in the linear protein are disordered, especially in comparison with the short circular construct, SH3circ−Δ, which forms a relatively rigid loop/turn (Figures 3A,B). This correlates with the observed higher order parameters for the “terminal” residues in SH3circ−Δ vs. SH3lin−wt. The observed chemical shift for Tyr190 HN in SH3circ−Δ suggests a higher tendency for hydrogen bonding to Tyr136, which further stabilizes contacts between the β5- and β1-strands. The circularization regions in SH3circ−GΔ and SH3circ−wt contain additional residues and, therefore, are more flexible than in SH3circ−Δ (Figures 3C,D). The structural similarity between the linear and circular SH3 constructs is also consistent with the fact (see above and Camarero et al., 2001b) that the ligand binding affinities of all circular SH3 constructs are comparable to that for linear SH3.


Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Schumann FH, Varadan R, Tayakuniyil PP, Grossman JH, Camarero JA, Fushman D - Front Chem (2015)

Comparison of the 3D structure of the backbone for the circular SH3 constructs with the solution and the (ligand-bound) crystal (Wu et al., 1995) structures for the linear domain. The solution structures are represented by the mean structure for each ensemble, colored green (SH3lin-wt), cyan (SH3circ-Δ), blue (SH3circ-GΔ), and yellow (SH3circ-wt). The crystal structure is colored red. These structures were superimposed using backbone heavy atoms for the core residues. The two panels represent two different orientations of the protein: (A) similar to that in Figure 1 and (B) view from the bottom. Elements or secondary structure and the termini are indicated. The drawings were made using InsightII.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389572&req=5

Figure 7: Comparison of the 3D structure of the backbone for the circular SH3 constructs with the solution and the (ligand-bound) crystal (Wu et al., 1995) structures for the linear domain. The solution structures are represented by the mean structure for each ensemble, colored green (SH3lin-wt), cyan (SH3circ-Δ), blue (SH3circ-GΔ), and yellow (SH3circ-wt). The crystal structure is colored red. These structures were superimposed using backbone heavy atoms for the core residues. The two panels represent two different orientations of the protein: (A) similar to that in Figure 1 and (B) view from the bottom. Elements or secondary structure and the termini are indicated. The drawings were made using InsightII.
Mentions: Our chemical shift data and structure calculations both indicate that structural perturbations in the SH3 domain caused by circularization are small. Figure 7 presents a superposition of the three-dimensional structures of all SH3 constructs studied here and of the crystal structure of linear SH3 in the bound form. The backbone fold is very similar in all these SH3 constructs; the pair-wise RMSDs between the mean NMR structures (for each NMR ensemble) are 0.8–1.1 Å and reduce to 0.3–0.6 Å if only core residues are considered (Table 3). As expected, the termini in the linear protein are disordered, especially in comparison with the short circular construct, SH3circ−Δ, which forms a relatively rigid loop/turn (Figures 3A,B). This correlates with the observed higher order parameters for the “terminal” residues in SH3circ−Δ vs. SH3lin−wt. The observed chemical shift for Tyr190 HN in SH3circ−Δ suggests a higher tendency for hydrogen bonding to Tyr136, which further stabilizes contacts between the β5- and β1-strands. The circularization regions in SH3circ−GΔ and SH3circ−wt contain additional residues and, therefore, are more flexible than in SH3circ−Δ (Figures 3C,D). The structural similarity between the linear and circular SH3 constructs is also consistent with the fact (see above and Camarero et al., 2001b) that the ligand binding affinities of all circular SH3 constructs are comparable to that for linear SH3.

Bottom Line: On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site.In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work.These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland College Park, MD, USA.

ABSTRACT
Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

No MeSH data available.


Related in: MedlinePlus