81 research outputs found
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Divide and Conquer: High Resolution Structural Information on TRP Channel Fragments
Understanding how proteins facilitate signaling and substrate transport across biological membranes is an important frontier of structural biology. Membrane proteins are the doors and windows of cells: many membrane proteins are gates of entry into or exit from cells or cellular compartments, and others allow cells to sense their environment. One important multifunctional family of membrane proteins is the transient receptor potential (TRP) family of ion channels. TRP channels have recently been the subject of multiple structural analyses, both low resolution electron microscopy studies (reviewed by Moiseenkova-Bell and Wensel in this issue [p. 239]) and the divide and conquer approach of determining high resolution crystal structures of channel fragments, reviewed here.Molecular and Cellular Biolog
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TRP Channels Entering the Structural Era
Transient receptor potential (TRP) channels are important in many neuronal and non-neuronal physiological processes. The past 2 years have seen much progress in the use of structural biology techniques to elucidate molecular mechanisms of TRP channel gating and regulation. Two approaches have proven fruitful: (i) a divide-and-conquer strategy has provided high-resolution structural details of TRP channel fragments although it fails to explain how these fragments are integrated in the full channel; and (ii) electron microscopy of entire TRP channels has yielded low-resolution images that provide a basis for testable models of TRP channel architecture. The results of each approach, summarized in this review, provide a preview of what the future holds in TRP channel structural biology.Molecular and Cellular Biolog
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Antigen Processing and Presentation: TAPping into ABC Transporters
Adaptive, cell-mediated immunity involves the presentation of antigenic peptides on
class I MHC molecules at the cell surface. This requires an ABC transporter associated
with antigen processing (TAP) to transport antigenic peptides generated in the cytosol
into the endoplasmic reticulum (ER) for loading onto class I MHC. Recent crystal
structures of bacterial ABC transporters suggest how the transmembrane domains of TAP
form a peptide-binding cavity that acquires peptides from the cytosol, and following
ATP-induced conformational changes, the peptide-binding cavity closes to the cytosol
and instead opens to the ER lumen for peptide release. Extensive biochemical studies
show how transport is driven by ATP binding and hydrolysis on an asymmetric pair of
cytosolic nucleotide-binding domains, which are physically coupled to the peptide binding
site to propagate conformational changes through the protein.Molecular and Cellular Biolog
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Distinct Properties of -Calmodulin Binding to N- and C-Terminal Regulatory Regions of the TRPV1 Channel
Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM in complex with the TRPV1-CT segment, determined to resolution. The two lobes of wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of , respectively. This structure is similar to canonical -peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity -binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the -binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the -binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.Molecular and Cellular Biolog
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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.Molecular and Cellular Biolog
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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.Molecular and Cellular Biolog
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What do we know about the transient receptor potential vanilloid 2 (TRPV2) ion channel?
Transient receptor potential (TRP) ion channels are emerging as a new set of membrane proteins involved in a vast array of cellular processes and regulated by a large number of physical and chemical stimuli, which involves them with sensory cell physiology. The vanilloid TRP subfamily (TRPV) named after the vanilloid receptor 1 (TRPV1) consists of six members, and at least four of them (TRPV1–TRPV4) have been related to thermal sensation. One of the least characterized members of the TRP subfamily is TRPV2. Although initially characterized as a noxious heat sensor, TRPV2 now seems to have little to do with temperature sensing but a much more complex physiological profile. Here we review the available information and research progress on the structure, physiology and pharmacology of TRPV2 in an attempt to shed some light on the physiological and pharmacological deorphanization of TRPV2.Molecular and Cellular Biolog
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. These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a -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 removal owing to increased flexure of -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.Molecular and Cellular Biolog
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A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15
Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca2+-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca2+-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca2+-binding linker regions: canonical EC4-5 linker binding three Ca2+ ions, and non-canonical EC3-4 linker binding only two Ca2+ ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca2+-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.Molecular and Cellular Biolog
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