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• 1. [Article] Allosteric Mechanism of Water Channel Gating by Ca2+–calmodulin

  Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important ...

  Citation × Citation Citation Abstract Copy Title: Allosteric Mechanism of Water Channel Gating by Ca2+–calmodulin Author: Reichow, Steve, Clemens, Daniel M., Freites, J. Alfredo, Németh-Cahalan, Karin L., Heyden, Matthias, Tobias, Douglas J., Hall, James E., Gonen, Tamir Year: 2013 Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. Title: Allosteric Mechanism of Water Channel Gating by Ca2+–calmodulin Author: Reichow, Steve, Clemens, Daniel M., Freites, J. Alfredo, Németh-Cahalan, Karin L., Heyden, Matthias, Tobias, Douglas J., Hall, James E., Gonen, Tamir Year: 2013 Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. Close

• 2. [Article] Non-canonical Binding of Calmodulin to Aquaporin-0: Implications for Channel Regulation

  Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homo-tetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) ...

  Citation × Citation Citation Abstract Copy Title: Non-canonical Binding of Calmodulin to Aquaporin-0: Implications for Channel Regulation Author: Reichow, Steve, Gonen, Tamir Year: 2008 Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homo-tetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show CaM binds the AQP0 C-terminal domain in a calcium dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a non-canonical fashion, suggesting a form of co-operativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0’s CaM binding domain inhibits the AQP0/ CaM interaction suggesting a temporal regulatory mechanism for complex formation. Title: Non-canonical Binding of Calmodulin to Aquaporin-0: Implications for Channel Regulation Author: Reichow, Steve, Gonen, Tamir Year: 2008 Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homo-tetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show CaM binds the AQP0 C-terminal domain in a calcium dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a non-canonical fashion, suggesting a form of co-operativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0’s CaM binding domain inhibits the AQP0/ CaM interaction suggesting a temporal regulatory mechanism for complex formation. Close

• 3. [Article] Cooperative Interaction of Transcription Termination Factors with the RNA Polymerase II C-terminal Domain

  Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTDinteracting domains ...

  Citation × Citation Citation Abstract Copy Title: Cooperative Interaction of Transcription Termination Factors with the RNA Polymerase II C-terminal Domain Author: Lunde, Bradley M., Reichow, Steve, Kim, Minkyu, Suh, Hyunsuk, Leeper, Thomas C., Yang, Fan, Mutschler, Hannes, Buratowski, Stephen, Meinhart, Anton, Varani, Gabriele Year: 2010 Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTDinteracting domains that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CTD-interacting domains of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single amino acid mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′-end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Serine 2 phosphorylation density is highest. Title: Cooperative Interaction of Transcription Termination Factors with the RNA Polymerase II C-terminal Domain Author: Lunde, Bradley M., Reichow, Steve, Kim, Minkyu, Suh, Hyunsuk, Leeper, Thomas C., Yang, Fan, Mutschler, Hannes, Buratowski, Stephen, Meinhart, Anton, Varani, Gabriele Year: 2010 Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTDinteracting domains that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CTD-interacting domains of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single amino acid mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′-end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Serine 2 phosphorylation density is highest. Close

• 4. [Article] AKAP2 Anchors PKA with Aquaporin-0 to Support Ocular Lens Transparency

  A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and ...

  Citation × Citation Citation Abstract Copy Title: AKAP2 Anchors PKA with Aquaporin-0 to Support Ocular Lens Transparency Author: Gold, Matthew G., Reichow, Steve, O'Neill, Susan E., Weisbrod, Chad R., Langeberg, Lorene K., Bruce, James E., Gonen, Tamir, Scott, John D. Year: 2011 A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a calmodulin (CaM)-binding domain of AQP0. The additional negative charge introduced by phosphoserine 235 perturbs electrostatic interactions between AQP0 and CaM to favour water influx through the channel. In isolated mouse lenses, displacement of PKA from the AKAP2–AQP0 channel complex promotes cortical cataracts as characterized by severe opacities and cellular damage. Thus, anchored PKA modulation of AQP0 is a homeostatic mechanism that must be physically intact to preserve lens transparency. Title: AKAP2 Anchors PKA with Aquaporin-0 to Support Ocular Lens Transparency Author: Gold, Matthew G., Reichow, Steve, O'Neill, Susan E., Weisbrod, Chad R., Langeberg, Lorene K., Bruce, James E., Gonen, Tamir, Scott, John D. Year: 2011 A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a calmodulin (CaM)-binding domain of AQP0. The additional negative charge introduced by phosphoserine 235 perturbs electrostatic interactions between AQP0 and CaM to favour water influx through the channel. In isolated mouse lenses, displacement of PKA from the AKAP2–AQP0 channel complex promotes cortical cataracts as characterized by severe opacities and cellular damage. Thus, anchored PKA modulation of AQP0 is a homeostatic mechanism that must be physically intact to preserve lens transparency. Close