Marcos Sotomayor

Single-Molecule Experiments in Vitro and in Silico

Single-molecule force experiments in vitro enable the characterization of the mechanical response of biological matter at the nanometer scale. However, they do not reveal the molecular mechanisms underlying mechanical function. These can only be readily studied through molecular dynamics simulations of atomic structural models: “in silico” (by computer analysis) single-molecule experiments. Steered molecular dynamics simulations, in which external forces are used to explore the response and function of macromolecules, have become a powerful tool complementing and guiding in vitro single-molecule experiments. The insights provided by in silico experiments are illustrated here through a review of recent research in three areas of protein mechanics: elasticity of the muscle protein titin and the extracellular matrix protein fibronectin; linker-mediated elasticity of the cytoskeleton protein spectrin; and elasticity of ankyrin repeats, a protein module found ubiquitously in cells but with an as-yet unclear function.

Molecular dynamics simulations of membrane channels and transporters.

Membrane transport constitutes one of the most fundamental processes in all living cells with proteins as major players. Proteins as channels provide highly selective diffusive pathways gated by environmental factors, and as transporters furnish directed, energetically uphill transport consuming energy. X-ray crystallography of channels and transporters furnishes a rapidly growing number of atomic resolution structures, permitting molecular dynamics (MD) simulations to reveal the physical mechanisms underlying channel and transporter function. Ever increasing computational power today permits simulations stretching up to 1 micros, that is, to physiologically relevant time scales. Membrane protein simulations presently focus on ion channels, on aquaporins, on protein-conducting channels, as well as on various transporters. In this review we summarize recent developments in this rapidly evolving field.

A structural mechanism for MscS gating in lipid bilayers.

The mechanosensitive channel of small conductance (MscS) is a key determinant in the prokaryotic response to osmotic challenges. We determined the structural rearrangements associated with MscS activation in membranes, using functorial measurements, electron paramagnetic resonance spectroscopy, and computational analyses. MscS was trapped in its open conformation after the transbilayer pressure profile was modified through the asymmetric incorporation of lysophospholipids. The transition from the closed to the open state is accompanied by the downward tilting of the transmembrane TM1-TM2 hairpin and by the expansion, tilt, and rotation of the TM3 helices. These movements expand the permeation pathway, leading to an increase in accessibility to water around TM3. Our open MscS model is compatible with single-channel conductance measurements and supports the notion that helix tilting is associated with efficient pore widening in mechanosensitive channels.

Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction

Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a Ca2+-dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15–cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex is shown to become unstable in response to Ca2+ removal owing to increased flexure of Ca2+-free cadherin repeats. Finally, we use structures and biochemical measurements to study the molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis, neural connectivity and cancer.

Structural Determinants of Cadherin-23 Function in Hearing and Deafness

The hair-cell tip link, a fine filament directly conveying force to mechanosensitive transduction channels, is composed of two proteins, protocadherin-15 and cadherin-23, whose mutation causes deafness. However, their molecular structure, elasticity, and deafness-related structural defects are unknown. We present crystal structures of the first and second extracellular cadherin repeats of cadherin-23. Overall, structures show typical cadherin folds, but reveal an elongated N terminus that precludes classical cadherin interactions and contributes to an N-terminal Ca(2+)-binding site. The deafness mutation D101G, in the linker region between the repeats, causes a slight bend between repeats and decreases Ca(2+) affinity. Molecular dynamics simulations suggest that cadherin-23 repeats are stiff and that either removing Ca(2+) or mutating Ca(2+)-binding residues reduces rigidity and unfolding strength. The structures define an uncharacterized cadherin family and, with simulations, suggest mechanisms underlying inherited deafness and how cadherin-23 may bind with itself and with protocadherin-15 to form the tip link.

Constant electric field simulations of the membrane potential illustrated with simple systems

Advances in modern computational methods and technology make it possible to carry out extensive molecular dynamics simulations of complex membrane proteins based on detailed atomic models. The ultimate goal of such detailed simulations is to produce trajectories in which the behavior of the system is as realistic as possible. A critical aspect that requires consideration in the case of biological membrane systems is the existence of a net electric potential difference across the membrane. For meaningful computations, it is important to have well validated methodologies for incorporating the latter in molecular dynamics simulations. A widely used treatment of the membrane potential in molecular dynamics consists of applying an external uniform electric field E perpendicular to the membrane. The field acts on all charged particles throughout the simulated system, and the resulting applied membrane potential V is equal to the applied electric field times the length of the periodic cell in the direction perpendicular to the membrane. A series of test simulations based on simple membrane-slab models are carried out to clarify the consequences of the applied field. These illustrative tests demonstrate that the constant-field method is a simple and valid approach for accounting for the membrane potential in molecular dynamics studies of biomolecular systems. This article is part of a Special Issue entitled: Membrane protein structure and function.

Three Dimensional Architecture of Membrane-Embedded MscS in the Closed Conformation

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in Escherichia coli. MscS opens as a result of membrane tension changes, thereby releasing small solutes and effectively acting as an osmotic safety valve. Both the functional state depicted by its crystal structure and its gating mechanism remain unclear. Here, we combine site-directed spin labeling, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations with novel energy restraints based on experimental electron paramagnetic resonance data to investigate the native transmembrane (TM) and periplasmic molecular architecture of closed MscS in a lipid bilayer. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, characterized by a realignment of the TM segments towards the normal of the membrane. The previously unresolved NH(2)-terminus forms a short helical hairpin capping the extracellular ends of TM1 and TM2 and is in close interaction with the bilayer interface. The present three-dimensional model of membrane-embedded MscS in the closed state represents a key step in determining the molecular mechanism of MscS gating.

Hearing: Tightrope act

A component of the ‘tip link’ that conveys tension to mechanically sensitive ion channels in the inner ear has been identified. The finding raises new questions about elastic elements in our hearing apparatus.

Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.

Sorting Out a Promiscuous Superfamily: Towards Cadherin Connectomics

Members of the cadherin superfamily of proteins are involved in diverse biological processes such as morphogenesis, sound transduction, and neuronal connectivity. Key to cadherin function is their extracellular domain containing cadherin repeats, which can mediate interactions involved in adhesion and cell signaling. Recent cellular, biochemical, and structural studies have revealed that physical interaction among cadherins is more complex than originally thought. Here we review work on new cadherin complexes and discuss how the classification of the mammalian family can be used to search for additional cadherin-interacting partners. We also highlight some of the challenges in cadherin research; namely, the characterization of a cadherin connectome in biochemical and structural terms, as well as the elucidation of molecular mechanisms underlying the functional diversity of nonclassical cadherins in vivo.