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Substrate induced population shifts and stochastic gating in the PBCV-1 mRNA capping enzyme.

Swift RV, McCammon JA - J. Am. Chem. Soc. (2009)

Bottom Line: Our results show that binding efficiency is a function of conformation but that isomerization between efficient and inefficient binding conformations does not impact the substrate association rate.Additionally, we show that conformational flexibility alone is insufficient to explain single stranded mRNA specificity.While our results are specific to the PBCV-1 mRNA capping enzyme, they provide a useful context within which the substrate binding behavior of similarly structured enzymes or proteins may be considered.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Center for Theoretical Biological Physics, University of California at San Diego, La Jolla, California, 92039-0365, USA. rswift@mccammon.ucsd.edu

ABSTRACT
The 317 residue PBCV-1 mRNA capping enzyme catalyzes the second enzymatic reaction in the formation of an N-7-methyl-GMP cap on the 5'-end of the nascent mRNA. It is composed of two globular domains bound by a short flexible peptide linker, which have been shown to undergo opening and closing events. The small size and experimentally demonstrated domain mobility make the PBCV-1 capping enzyme an ideally suited model system to explore domain mobility in context of substrate binding. Here, we specifically address the following four questions: (1) How does substrate binding affect relative domain mobility: is the system better described by an induced fit or population shift mechanism? (2) What are the gross characteristics of a conformation capable of binding substrate? (3) Does "domain gating" of the active site affect the rate of substrate binding? (4) Does the magnitude of receptor conformational fluctuations confer substrate specificity by sterically occluding molecules of a particular size or geometry? We answer these questions using a combination of theory, Brownian dynamics, and molecular dynamics. Our results show that binding efficiency is a function of conformation but that isomerization between efficient and inefficient binding conformations does not impact the substrate association rate. Additionally, we show that conformational flexibility alone is insufficient to explain single stranded mRNA specificity. While our results are specific to the PBCV-1 mRNA capping enzyme, they provide a useful context within which the substrate binding behavior of similarly structured enzymes or proteins may be considered.

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Conformational distributions in the apo and holo states: Probability distribution functions (PDFs) of the distance separating the centers of mass of the OB and nucleotidyltransferase domains in the MD simulations. Xc and Xo indicate the locations of the closed and open crystal structures, respectively. The holo distribution is represented by a dashed line and the apo distribution by a solid line. Structures of selected conformations are labeled with the distance separating the centers of mass of their OB and nucleotidyltransferase domains. The three lower structures are representatives of the apo simulation while the single upper structure is a representative of the holo simulations. Coloring is the same as in Figure 1.
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fig2: Conformational distributions in the apo and holo states: Probability distribution functions (PDFs) of the distance separating the centers of mass of the OB and nucleotidyltransferase domains in the MD simulations. Xc and Xo indicate the locations of the closed and open crystal structures, respectively. The holo distribution is represented by a dashed line and the apo distribution by a solid line. Structures of selected conformations are labeled with the distance separating the centers of mass of their OB and nucleotidyltransferase domains. The three lower structures are representatives of the apo simulation while the single upper structure is a representative of the holo simulations. Coloring is the same as in Figure 1.

Mentions: In order to determine whether GTP binding by the PBCV-1 capping enzyme is better described by the induced fit or the population shift model of substrate binding, we examine the distribution of conformations of the OB domain relative to the nucleotidyltransferase domain. As we are only interested in the relative motion of the OB domain, we neglect internal domain fluctuations and approximate the OB domain as a rigid body. This approximation is supported by earlier work in which OB-domain secondary structure was retained during isomerization from an open to a closed conformation.(19) By using the rigid body approximation, the position and orientation of the OB domain is described by three positional and three orientational degrees of freedom that are measured with respect to a coordinate system whose origin we center on the center of mass of the nucleotidyltransferase domain. Furthermore, relative to the large fluctuations between the centers of mass of the OB and nucleotidyltransferase domains observed during simulations of the apo state, fluctuations along the two orthogonal positional degrees of freedom, as well as fluctuations around the three orientational degrees of freedom, were modest. This observation is apparent in the three representative conformations from the apo state simulations shown in Figure 2. As fluctuations between the centers of mass of the OB and nucleotidyltransferase domains constitute the preponderance of relative domain motion, we approximate the distribution of conformations of the OB domain relative to the nucleotidyltransferase domain by the distribution of distances separating their centers of mass. The probability distribution functions (PDFs) of the distance separating the centers of mass of the domains in the apo and holo trajectories are reported in Figure 2. In the apo state, the two domains span a range of conformations and exhibit a bimodal distribution. Conversely, in the holo state, the distribution is localized around a single conformation that resembles the closed, holo crystal structure.


Substrate induced population shifts and stochastic gating in the PBCV-1 mRNA capping enzyme.

Swift RV, McCammon JA - J. Am. Chem. Soc. (2009)

Conformational distributions in the apo and holo states: Probability distribution functions (PDFs) of the distance separating the centers of mass of the OB and nucleotidyltransferase domains in the MD simulations. Xc and Xo indicate the locations of the closed and open crystal structures, respectively. The holo distribution is represented by a dashed line and the apo distribution by a solid line. Structures of selected conformations are labeled with the distance separating the centers of mass of their OB and nucleotidyltransferase domains. The three lower structures are representatives of the apo simulation while the single upper structure is a representative of the holo simulations. Coloring is the same as in Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837470&req=5

fig2: Conformational distributions in the apo and holo states: Probability distribution functions (PDFs) of the distance separating the centers of mass of the OB and nucleotidyltransferase domains in the MD simulations. Xc and Xo indicate the locations of the closed and open crystal structures, respectively. The holo distribution is represented by a dashed line and the apo distribution by a solid line. Structures of selected conformations are labeled with the distance separating the centers of mass of their OB and nucleotidyltransferase domains. The three lower structures are representatives of the apo simulation while the single upper structure is a representative of the holo simulations. Coloring is the same as in Figure 1.
Mentions: In order to determine whether GTP binding by the PBCV-1 capping enzyme is better described by the induced fit or the population shift model of substrate binding, we examine the distribution of conformations of the OB domain relative to the nucleotidyltransferase domain. As we are only interested in the relative motion of the OB domain, we neglect internal domain fluctuations and approximate the OB domain as a rigid body. This approximation is supported by earlier work in which OB-domain secondary structure was retained during isomerization from an open to a closed conformation.(19) By using the rigid body approximation, the position and orientation of the OB domain is described by three positional and three orientational degrees of freedom that are measured with respect to a coordinate system whose origin we center on the center of mass of the nucleotidyltransferase domain. Furthermore, relative to the large fluctuations between the centers of mass of the OB and nucleotidyltransferase domains observed during simulations of the apo state, fluctuations along the two orthogonal positional degrees of freedom, as well as fluctuations around the three orientational degrees of freedom, were modest. This observation is apparent in the three representative conformations from the apo state simulations shown in Figure 2. As fluctuations between the centers of mass of the OB and nucleotidyltransferase domains constitute the preponderance of relative domain motion, we approximate the distribution of conformations of the OB domain relative to the nucleotidyltransferase domain by the distribution of distances separating their centers of mass. The probability distribution functions (PDFs) of the distance separating the centers of mass of the domains in the apo and holo trajectories are reported in Figure 2. In the apo state, the two domains span a range of conformations and exhibit a bimodal distribution. Conversely, in the holo state, the distribution is localized around a single conformation that resembles the closed, holo crystal structure.

Bottom Line: Our results show that binding efficiency is a function of conformation but that isomerization between efficient and inefficient binding conformations does not impact the substrate association rate.Additionally, we show that conformational flexibility alone is insufficient to explain single stranded mRNA specificity.While our results are specific to the PBCV-1 mRNA capping enzyme, they provide a useful context within which the substrate binding behavior of similarly structured enzymes or proteins may be considered.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Center for Theoretical Biological Physics, University of California at San Diego, La Jolla, California, 92039-0365, USA. rswift@mccammon.ucsd.edu

ABSTRACT
The 317 residue PBCV-1 mRNA capping enzyme catalyzes the second enzymatic reaction in the formation of an N-7-methyl-GMP cap on the 5'-end of the nascent mRNA. It is composed of two globular domains bound by a short flexible peptide linker, which have been shown to undergo opening and closing events. The small size and experimentally demonstrated domain mobility make the PBCV-1 capping enzyme an ideally suited model system to explore domain mobility in context of substrate binding. Here, we specifically address the following four questions: (1) How does substrate binding affect relative domain mobility: is the system better described by an induced fit or population shift mechanism? (2) What are the gross characteristics of a conformation capable of binding substrate? (3) Does "domain gating" of the active site affect the rate of substrate binding? (4) Does the magnitude of receptor conformational fluctuations confer substrate specificity by sterically occluding molecules of a particular size or geometry? We answer these questions using a combination of theory, Brownian dynamics, and molecular dynamics. Our results show that binding efficiency is a function of conformation but that isomerization between efficient and inefficient binding conformations does not impact the substrate association rate. Additionally, we show that conformational flexibility alone is insufficient to explain single stranded mRNA specificity. While our results are specific to the PBCV-1 mRNA capping enzyme, they provide a useful context within which the substrate binding behavior of similarly structured enzymes or proteins may be considered.

Show MeSH
Related in: MedlinePlus