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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

The ensembles of 20 lowest-target-function structures (backbone) for the linear (A) and circular SH3 constructs: (B) SH3circ-Δ, (C) SH3circ-GΔ, and (D) SH3circ-wt. A cartoon representation of the 3D structure of the linear N-terminal SH3 domain of c-Crk is shown in (E). For the circular constructs the arrows indicate the location of the cyclization loop. Colored red are those residues exhibiting conformational exchange in the submillisecond time scale (cf. Figures 5, 6). The figure was prepared using MolMol (Koradi et al., 1996).
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Figure 1: The ensembles of 20 lowest-target-function structures (backbone) for the linear (A) and circular SH3 constructs: (B) SH3circ-Δ, (C) SH3circ-GΔ, and (D) SH3circ-wt. A cartoon representation of the 3D structure of the linear N-terminal SH3 domain of c-Crk is shown in (E). For the circular constructs the arrows indicate the location of the cyclization loop. Colored red are those residues exhibiting conformational exchange in the submillisecond time scale (cf. Figures 5, 6). The figure was prepared using MolMol (Koradi et al., 1996).

Mentions: As a model for these studies we have selected the N-terminal SH3 domain (57 residues long) of the adapter protein c-Crk (Knudsen et al., 1994). The structure of the free isolated SH3 domain was not available. According to the crystal structure of the SH3 domain complexed with a C3G-derived proline-rich peptide (Knudsen et al., 1994), the protein adopts a compact fold featuring five β-strands and a short 310-helix (Wu et al., 1995) (see Figure 1E). The native/folded state of SH3 is stabilized by a network of electrostatic and hydrophobic interactions. Specifically, the salt bridge between the side chains of residues Glu135 and Lys164 has been shown to be essential for the stability of the SH3 fold (Camarero et al., 2001b). Although Glu135 does not belong to the secondary structure of the protein, its deletion destabilizes the SH3 structure, resulting in a partially unfolded protein (Camarero et al., 2001b) with a ten-fold weaker affinity for the ligand. The juxtaposed N- and C-termini make this SH3 domain a favorable target for circularization.


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)

The ensembles of 20 lowest-target-function structures (backbone) for the linear (A) and circular SH3 constructs: (B) SH3circ-Δ, (C) SH3circ-GΔ, and (D) SH3circ-wt. A cartoon representation of the 3D structure of the linear N-terminal SH3 domain of c-Crk is shown in (E). For the circular constructs the arrows indicate the location of the cyclization loop. Colored red are those residues exhibiting conformational exchange in the submillisecond time scale (cf. Figures 5, 6). The figure was prepared using MolMol (Koradi et al., 1996).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The ensembles of 20 lowest-target-function structures (backbone) for the linear (A) and circular SH3 constructs: (B) SH3circ-Δ, (C) SH3circ-GΔ, and (D) SH3circ-wt. A cartoon representation of the 3D structure of the linear N-terminal SH3 domain of c-Crk is shown in (E). For the circular constructs the arrows indicate the location of the cyclization loop. Colored red are those residues exhibiting conformational exchange in the submillisecond time scale (cf. Figures 5, 6). The figure was prepared using MolMol (Koradi et al., 1996).
Mentions: As a model for these studies we have selected the N-terminal SH3 domain (57 residues long) of the adapter protein c-Crk (Knudsen et al., 1994). The structure of the free isolated SH3 domain was not available. According to the crystal structure of the SH3 domain complexed with a C3G-derived proline-rich peptide (Knudsen et al., 1994), the protein adopts a compact fold featuring five β-strands and a short 310-helix (Wu et al., 1995) (see Figure 1E). The native/folded state of SH3 is stabilized by a network of electrostatic and hydrophobic interactions. Specifically, the salt bridge between the side chains of residues Glu135 and Lys164 has been shown to be essential for the stability of the SH3 fold (Camarero et al., 2001b). Although Glu135 does not belong to the secondary structure of the protein, its deletion destabilizes the SH3 structure, resulting in a partially unfolded protein (Camarero et al., 2001b) with a ten-fold weaker affinity for the ligand. The juxtaposed N- and C-termini make this SH3 domain a favorable target for circularization.

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