Insights into calcium transport enhance our understanding of muscle function

21-04-2020

The mechanisms of muscle function are not completely understood on the molecular level. Research performed at beamline ID09 has revealed how a protein that regulates muscle relaxation undergoes structural change when pumping calcium from the muscle cell to its storage.

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To perform reactions essential to Life, proteins need to change their 3D structures. This inherent flexibility has been encoded into the amino acid sequence of the protein during evolution. To understand protein function on the molecular level, it is necessary to map how the protein components move, that is how the protein transitions between different intermediate states.

Some intermediate states can be stabilised, which enables structural determination, but most states are unfortunately not stable enough to enable such experiments. By monitoring the reaction using fast X-ray pulses directly in the natural environment, it is possible to track the development of intermediate states without the need for artificial stabilisation methods, such as in protein crystallography.

The pump and probe setup at beamline ID09 is ideally suited to recording subtle structural changes in complex biological macromolecules. A laser pulse is typically used to trigger the protein reaction followed by X-ray probe pulses capable of delivering 1010 photons onto the sample during a 100-picosecond pulse. Traditional biological targets have been proteins that are light-sensitive, either by nature or by design because a laser is used to initiate the reaction.

In this new study, the reaction has been initiated by laser-induced release of ATP from a light-sensitive inactive form of ATP. A large number of light-sensitive substances such as neurotransmitters, metabolites, and ions could enable similar experiments for a range of important protein targets.

The protein investigated in this study regulates relaxation of skeletal muscle and is also of importance for, e.g., normal heart functionality. To allow for muscles to relax, the calcium used during contraction needs to be transported back to the sarcoplasmic reticulum, which is a tube-like system that surrounds the muscle cell.

Calcium cannot pass through the sarcoplasmic reticulum membrane unaided, but needs active pumping by the SERCA protein. The pumping process demands energy and the SERCA protein will not start without access to the energy carrier ATP. Despite several known intermediate structures, trapped by using ATP analogues and others, structural information from the critical transition when the protein transports calcium back to the sarcoplasmic reticulum storage to allow the muscle to relax, is still elusive.

In a series of experiments at beamline ID09, time-resolved X-ray data was collected directly on the biological sarcoplasmic reticulum membranes, where up to 90% of the protein content consists of SERCA proteins. However, because the measurements are performed on a solution that consists of many circular membrane fractions that move freely relative to each other, the retrieved structural information will be of low resolution, i.e. without details. Today, the path leading to a detailed molecular view of the registered reaction, so-called structural refinement, is far from standardised.

In this study, by exploiting state-of-the-art supercomputers and algorithms, it has been possible to identify two structures of SERCA intermediate states (Figure 1). In the first observed state at 1.5 milliseconds the protein had closed around the ATP molecule and taken up calcium from the muscle cell. Then, at 13 milliseconds, a state with a so far unknown structure, was observed that represents the protein in the moment before releasing calcium back to its sarcoplasmic reticulum storage compartment (Figure 2).

The resting state and the two transient intermediates superimposed onto crystal structures of the corresponding closest states.

Figure 1. The resting state and the two transient intermediates identified in the study superimposed onto crystal structures of the corresponding closest states (transparent white).

The observation of new protein structures helps to understand the underlying molecular mechanisms for muscle function, which is crucial to understanding diseases such as heart disease.

The location of a domain that controls the switch between states open to the muscle cell cytoplasm (E1, depicted in blue) and the outside storage space (E2, depicted in magenta).

Figure 2. The location of a domain that controls the switch between states open to the muscle cell cytoplasm (E1, depicted in blue) and the outside storage space (E2, depicted in magenta). In the 13-millisecond intermediate, this domain is located in-between the E1 and E2 positions.

In conclusion, time-resolved X-ray scattering experiments not only enable structural determination of instable intermediates, but also deliver these structures with a time stamp. In this way, it becomes possible to determine how different intermediate states develop over time and to study how their reaction kinetics differ with environmental cues, such as pH, temperature, and chemical composition, or under conditions that are characteristic of, e.g., mutation-induced diseases.

 

Principal publication and authors

Ravishankar, H. (a), Pedersen, M.N. (b), Eklund, M., Sitsel, A. (c), Li, C. (d), Duelli, A. (e), Levantino, M. (b), Wulff, M. (b), Barth, A. (d), Olesen, C. (c), Nissen, P. (c), Andersson, M. (a), Tracking Ca2+ ATPase intermediates in real-time by X-ray solution scattering, Science Advances 6, p.eaaz0981 (2020); DOI: 10.1126/sciadv.aaz0981.
(a) Umeå University, Umeå (Sweden)
(b) ESRF
(c) Aarhus University (Denmark)
(d) Stockholm University (Sweden)
(e) University of Copenhagen (Denmark)