A dynamically tuned double‐stranded RNA binding mechanism for the activation of antiviral kinase PKR

Abstract

A key step in the activation of interferon-inducible antiviral kinase PKR involves differential binding of viral double-stranded RNA (dsRNA) to its two structurally similar N-terminal dsRNA binding motifs, dsRBM1 and dsRBM2. We show here, using NMR spectroscopy, that dsRBM1 with higher RNA binding activity exhibits signi®cant motional¯exibility on a millisecond timescale as compared with dsRBM2 with lower RNA binding activity. We further show that dsRBM2, but not dsRBM1, speci®cally interacts with the C-terminal kinase domain. These results suggest a dynamically tuned dsRNA binding mechanism for PKR activation, where motionally more¯exible dsRBM1 anchors to dsRNA, thereby inducing a cooperative RNA binding for dsRBM2 to expose the kinase domain.

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double‐stranded RNA binding domain of protein kinase R

“…Thus, although we can not make any direct comparison between the flexibility of loops 2 and 7, we can conclude that loop 7 is definitely very flexible. This partially supports earlier conclusion that loop 7 is involved in dsRNA‐binding 1…”

“…4(B, C)]. This is similar to the observations made earlier based on the 15 N relaxation data 1. This type of higher flexibility seen in the linker region compared to the two motifs suggests that the two motifs reorient independently as in the case of calmodulin 29.…”

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double‐stranded RNA binding domain of protein kinase R

“…Thus, although we can not make any direct comparison between the flexibility of loops 2 and 7, we can conclude that loop 7 is definitely very flexible. This partially supports earlier conclusion that loop 7 is involved in dsRNA‐binding 1…”

“…4(B, C)]. This is similar to the observations made earlier based on the 15 N relaxation data 1. This type of higher flexibility seen in the linker region compared to the two motifs suggests that the two motifs reorient independently as in the case of calmodulin 29.…”

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification

“…We have observed this type of simultaneous binding by the two dsRBMs to another PKR ligand that activates the enzyme ( 42 ). These observations are consistent with a previous proposal that activation requires enough RNA to bind both of PKR's dsRBMs ( 29 ). Furthermore, given that PKR's dsRBMs show little sequence specificity, a similar complex can be envisioned with PKR's dsRBMs bound at the opposite end of the duplex.…”

Domain Stabilities in Protein Kinase R (PKR): Evidence for Weak Interdomain Interactions

“…Analysis of the thermodynamic stability of PKR and its constituent domains by equilibrium chemical denaturation and limited proteolysis experiments indicates limited interaction between domains. Consistent with previous NMR evidence that dsRBM1 and dsRBM2 do not interact , , we find that their folding energetics are noncooperative and the linker connecting them is accessible to cleavage by trypsin. Although the overall structures of the two domains are quite similar, with a backbone rmsd of 2 Å , dsRBM2 is about 1.6 kcal/mol more stable than dsRBM1.…”

“…PKR is specifically cleaved by caspases at D251 upon stimulation of apoptosis, resulting in enhanced phosphorylation of eIF2R and inhibition of protein synthesis (48). We also observe that the linker lying between dsRBM1 and dsRBM2 can be cleaved by trypsin at K79, consistent with previous evidence that this region is flexible and unstructured (9,10). Interestingly, these interdomain cleavage reactions are not appreciably affected upon binding ATP analogues or dsRNA activators.…”

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double‐stranded RNA binding domain of protein kinase R

“…Thus, although we can not make any direct comparison between the flexibility of loops 2 and 7, we can conclude that loop 7 is definitely very flexible. This partially supports earlier conclusion that loop 7 is involved in dsRNA‐binding 1…”

“…4(B, C)]. This is similar to the observations made earlier based on the 15 N relaxation data 1. This type of higher flexibility seen in the linker region compared to the two motifs suggests that the two motifs reorient independently as in the case of calmodulin 29.…”

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification

“…We have observed this type of simultaneous binding by the two dsRBMs to another PKR ligand that activates the enzyme ( 42 ). These observations are consistent with a previous proposal that activation requires enough RNA to bind both of PKR's dsRBMs ( 29 ). Furthermore, given that PKR's dsRBMs show little sequence specificity, a similar complex can be envisioned with PKR's dsRBMs bound at the opposite end of the duplex.…”

Domain Stabilities in Protein Kinase R (PKR): Evidence for Weak Interdomain Interactions

“…Analysis of the thermodynamic stability of PKR and its constituent domains by equilibrium chemical denaturation and limited proteolysis experiments indicates limited interaction between domains. Consistent with previous NMR evidence that dsRBM1 and dsRBM2 do not interact , , we find that their folding energetics are noncooperative and the linker connecting them is accessible to cleavage by trypsin. Although the overall structures of the two domains are quite similar, with a backbone rmsd of 2 Å , dsRBM2 is about 1.6 kcal/mol more stable than dsRBM1.…”

“…PKR is specifically cleaved by caspases at D251 upon stimulation of apoptosis, resulting in enhanced phosphorylation of eIF2R and inhibition of protein synthesis (48). We also observe that the linker lying between dsRBM1 and dsRBM2 can be cleaved by trypsin at K79, consistent with previous evidence that this region is flexible and unstructured (9,10). Interestingly, these interdomain cleavage reactions are not appreciably affected upon binding ATP analogues or dsRNA activators.…”

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double‐stranded RNA binding domain of protein kinase R

“…Thus, although we can not make any direct comparison between the flexibility of loops 2 and 7, we can conclude that loop 7 is definitely very flexible. This partially supports earlier conclusion that loop 7 is involved in dsRNA‐binding 1…”

“…4(B, C)]. This is similar to the observations made earlier based on the 15 N relaxation data 1. This type of higher flexibility seen in the linker region compared to the two motifs suggests that the two motifs reorient independently as in the case of calmodulin 29.…”

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification

“…We have observed this type of simultaneous binding by the two dsRBMs to another PKR ligand that activates the enzyme ( 42 ). These observations are consistent with a previous proposal that activation requires enough RNA to bind both of PKR's dsRBMs ( 29 ). Furthermore, given that PKR's dsRBMs show little sequence specificity, a similar complex can be envisioned with PKR's dsRBMs bound at the opposite end of the duplex.…”

Domain Stabilities in Protein Kinase R (PKR): Evidence for Weak Interdomain Interactions

“…Analysis of the thermodynamic stability of PKR and its constituent domains by equilibrium chemical denaturation and limited proteolysis experiments indicates limited interaction between domains. Consistent with previous NMR evidence that dsRBM1 and dsRBM2 do not interact , , we find that their folding energetics are noncooperative and the linker connecting them is accessible to cleavage by trypsin. Although the overall structures of the two domains are quite similar, with a backbone rmsd of 2 Å , dsRBM2 is about 1.6 kcal/mol more stable than dsRBM1.…”

“…PKR is specifically cleaved by caspases at D251 upon stimulation of apoptosis, resulting in enhanced phosphorylation of eIF2R and inhibition of protein synthesis (48). We also observe that the linker lying between dsRBM1 and dsRBM2 can be cleaved by trypsin at K79, consistent with previous evidence that this region is flexible and unstructured (9,10). Interestingly, these interdomain cleavage reactions are not appreciably affected upon binding ATP analogues or dsRNA activators.…”