Dataset Information

Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions.

ABSTRACT: The viral RNA-dependent RNA polymerases show a conserved structure where the fingers domain interacts with the top of the thumb domain to create a tunnel through which nucleotide triphosphates reach the active site. We have solved the crystal structures of poliovirus polymerase (3D(pol)) in complex with all four NTPs, showing that they all bind in a common pre-insertion site where the phosphate groups are not yet positioned over the active site. The NTPs interact with both the fingers and palm domains, forming bridging interactions that explain the increased thermal stability of 3D(pol) in the presence of NTPs. We have also examined the importance of the fingers-thumb domain interaction for the function and structural stability of 3D(pol). Results from thermal denaturation experiments using circular dichroism and 2-anilino-6-napthaline-sulfonate (ANS) fluorescence show that 3D(pol) has a melting temperature of only approximately 40 degrees C. NTP binding stabilizes the protein and increases the melting by 5-6 degrees C while mutations in the fingers-thumb domain interface destabilize the protein and reduce the melting point by as much as 6 degrees C. In particular, the burial of Phe30 and Phe34 from the tip of the index finger into a pocket at the top of the thumb and the presence of Trp403 on the thumb domain are key interactions required to maintain the structural integrity of the polymerase. The data suggest the fingers domain has significant conformational flexibility and exists in a highly dynamic molten globule state at physiological temperature. The role of the enclosed active site motif as a structural scaffold for constraining the fingers domain and accommodating conformational changes in 3D(pol) and other viral polymerases during the catalytic cycle is discussed.

SUBMITTER: Thompson AA

PROVIDER: S-EPMC1941708 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions.

Thompson Aaron A AA Albertini Rebecca A RA Peersen Olve B OB

Journal of molecular biology 20061201 5

The viral RNA-dependent RNA polymerases show a conserved structure where the fingers domain interacts with the top of the thumb domain to create a tunnel through which nucleotide triphosphates reach the active site. We have solved the crystal structures of poliovirus polymerase (3D(pol)) in complex with all four NTPs, showing that they all bind in a common pre-insertion site where the phosphate groups are not yet positioned over the active site. The NTPs interact with both the fingers and palm d ...[more]

PMID: 17223130

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Project description:Background:In humans, the thumb plays a crucial role in producing finger opposition movements. These movements form the basis of several activities of the hand. Hence these movements have been used to study phenomena like prehension, motor control, motor learning, etc. Although such tasks have been studied extensively, the relative contribution of the thumb vis-à-vis the fingers in finger opposition tasks is not well understood. In this study, we investigated the kinematics of thumb and fingers in a simple finger opposition task. Further, we quantified the relative contribution and the movement smoothness aspects and compared these between fingers and thumb. Methods:Eight, young healthy participants (four males and four females) were asked to perform a full finger to thumb opposition movement, where they were required to reach for different phalanges of the fingers. Position (X, Y and Z) of individual segments of the four fingers and the thumb were measured with reference to the wrist by a 16-sensor kinematics measurement system. Displacements and velocities were computed. An index, displacement ratio, that quantifies the relative contribution of thumb and fingers was computed from displacement data. Velocity data was used to quantify the smoothness of movement of thumb and fingers. Results:The Displacement Ratio showed that contribution of the thumb is higher than contribution of any other target finger or target phalanges, except for the distal phalanx of the index and middle fingers. Smoothness of movement of the thumb was higher than all the finger phalanges in all cases. Conclusion:We conclude that in the task considered (thumb opposition movements to different targets within the hand & fingers), the thumb made a greater relative contribution in terms of displacement ratio and also produced smoother movements. However, smoothness of thumb did not vary depending on the target. This suggests that the traditional notion of the thumb being a special digit when compared to other fingers is true at least for the opposition movements considered in this study.

Project description:BackgroundThe human hand plays a crucial role in accomplishing activities of daily living. The contribution of each finger in the human hand is remarkably unique in establishing object stabilization. According to the mechanical advantage hypothesis, the little finger tends to exert a greater normal force than the ring finger during a supination moment production task to stabilize the object. Similarly, during pronation, the index finger produces more normal force when compared with the middle finger. Hence, the central nervous system employs the peripheral fingers for torque generation to establish the equilibrium as they have a mechanical advantage of longer moment arms for normal force. In our study, we tested whether the mechanical advantage hypothesis is supported in a task in which the contribution of thumb was artificially reduced. We also computed the safety margin of the individual fingers and thumb.MethodologyFifteen participants used five-finger prismatic precision grip to hold a custom-built handle with a vertical railing on the thumb side. A slider platform was placed on the railing such that the thumb sensor could move either up or down. There were two experimental conditions. In the "Fixed" condition, the slider was mechanically fixed, and hence the thumb sensor could not move. In the "Free" condition, the slider platform on which the thumb sensor was placed could freely move. In both conditions, the instruction was to grasp and hold the handle (and the platform) in static equilibrium. We recorded tangential and normal forces of all the fingers.ResultsThe distribution of fingertip forces and moments changed depending on whether the thumb platform was movable (or not). In the free condition, the drop in the tangential force of thumb was counteracted by an increase in the normal force of the ring and little finger. Critically, the normal forces of the ring and little finger were statistically equivalent. The safety margin of the index and middle finger did not show a significant drop in the free condition when compared to fixed condition.ConclusionWe conclude that our results does not support the mechanical advantage hypothesis at least for the specific mechanical task considered in our study. In the free condition, the normal force of little finger was comparable to the normal force of the ring finger. Also, the safety margin of the thumb and ring finger increased to prevent slipping of the thumb platform and to maintain the handle in static equilibrium during the free condition. However, the rise in the safety margin of the ring finger was not compensated by a drop in the safety margin of the index and middle finger.

Dataset Information

Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions.

Publications

Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions.

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