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N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the brain are important for controlling neuronal excitability to promote synaptic plasticity. The cytoskeletal protein, α-actinin-1 (100 kDa, called ACTN1) binds to the cytosolic C0 domain of GluN1 (residues 841-865) that may play a role in the Ca 2+ -dependent desensitization of NMDAR channels. Mutations that disrupt NMDAR channel function are linked to Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for the C-terminal EF-hand domain of ACTN1 (residues 824-892, called ACTN_EF34) and ACTN_EF34 bound to the GluN1 C0 domain (BMRB numbers 52385 and 52386, respectively)., (© 2024. The Author(s).)

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5 R22A ) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5 WT ). Here, we present ITC, NMR, and functional analysis of GCAP5 R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5 R22A binds a total of 3 Ca 2+ , including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5 WT were not detected by ITC, suggesting that R22A may affect the binding of Ca 2+ to these sites. The NMR-derived structure of GCAP5 R22A is overall similar to that of GCAP5 WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5 R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5 R22A with ∼25% higher affinity compared to GCAP5 WT , consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

Retinal membrane guanylyl cyclases (RetGCs) in vertebrate rod and cone photoreceptors are activated by a family of neuronal Ca 2+ sensor proteins called guanylyl cyclase activating proteins (GCAP1-7). GCAP5 from zebrafish photoreceptors binds to RetGC and confers Ca 2+ /Fe 2+ -dependent regulation of RetGC enzymatic activity that promotes the recovery phase of visual phototransduction. We report NMR chemical shift assignments of GCAP5 with a R22A mutation (called GCAP5 R22A ) that abolishes protein dimerization and activates RetGC with 3-fold higher activity than that of wild type GCAP5 (BMRB No. 51,783)., (© 2023. The Author(s).)

Neuroplasticity and synaptic transmission in the brain are regulated by N-methyl-D-aspartate receptors (NMDARs) that consist of hetero-tetrameric combinations of the glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binds to the cytosolic C0 domain of GluN1 (residues 841-865) that may play a role in the Ca 2+ -dependent inactivation (CDI) of NMDAR channel activity. Dysregulation of NMDARs are linked to various neurological disorders, including Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. Here, we report complete NMR chemical shift assignments of Ca 2+ -saturated CaM bound to the GluN1 C0 domain of the human NMDAR (BMRB no. 51715)., (© 2023. The Author(s).)

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the neuronal post-synaptic membrane are important for controlling neuroplasticity and synaptic transmission in the brain. Calmodulin (CaM) binds to the cytosolic C0 domains of both GluN1 (residues 841-865) and GluN2 (residues 1004-1024) that may play a role in the Ca 2+ -dependent desensitization of NMDAR channels. Mutations that disrupt Ca 2+ -dependent desensitization of NMDARs are linked to Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for Ca 2+ -saturated CaM bound to the GluN2A C0 domain of NMDAR (BMRB no. 51821)., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The L-type Ca 2+ channel Ca V 1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs Ca V 1.2 open probability (Po) and Ca 2+ -dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca 2+ -saturated CaM species (Ca 2 /CaM) with Ca 2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca 2+ concentrations (50-100 nM) that constitutively preassociates with Ca V 1.2 to promote Po and CDI. We also present an NMR structure of a complex between the Ca V 1.2 IQ motif (residues 1644-1665) and Ca 2 /CaM 12' , a calmodulin mutant in which Ca 2+ binding to EF1 and EF2 is completely disabled. We found that the CaM 12' N-lobe does not interact with the IQ motif. The CaM 12' C-lobe bound two Ca 2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca 2+ binding to EF3 and EF4 in the CaM 34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca 2 /CaM species promotes Ca V 1.2 Po and CDI, identifying Ca 2 /CaM as an important mediator of Ca signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Retinal cyclic nucleotide-gated (CNG) ion channels bind to intracellular cGMP and mediate visual phototransduction in photoreceptor rod and cone cells. Retinal rod CNG channels form hetero-tetramers comprised of three CNGA1 and one CNGB1 protein subunits. Cone CNG channels are similar tetramers consisting of three CNGA3 and one CNGB3 subunits. Calmodulin (CaM) binds to two distinct sites (CaM1: residues 565-587 and CaM2: residues 1120-1147) within the cytosolic domains of rod CNGB1. The binding of Ca 2+ -bound CaM to CNGB1 promotes the Ca 2+ -induced desensitization of CNG channels in retinal rods that may be important for photoreceptor light adaptation. Mutations that affect Ca 2+ -dependent CNG channel function are responsible for inherited forms of blindness. In this review, we propose structural models of the rod CNG channel bound to CaM that suggest how CaM might cause channel desensitization and how dysregulation of the channel may lead to retinal disease.

Calmodulin (CaM) binds to the membrane-proximal cytosolic C-terminal domain of Ca V 1.2 (residues 1520-1669, CT(1520-1669)) and causes Ca 2+ -induced conformational changes that promote Ca 2+ -dependent channel inactivation (CDI). We report biophysical studies that probe the structural interaction between CT(1520-1669) and CaM. The recombinantly expressed CT(1520-1669) is insoluble, but can be solubilized in the presence of Ca 2+ -saturated CaM (Ca 4 /CaM), but not in the presence of Ca 2+ -free CaM (apoCaM). We show that half-calcified CaM (Ca 2 /CaM 12 ) forms a complex with CT(1520-1669) that is less soluble than CT(1520-1669) bound to Ca 4 /CaM. The NMR spectrum of CT(1520-1669) reveals spectral differences caused by the binding of Ca 2 /CaM 12 versus Ca 4 /CaM, suggesting that the binding of Ca 2+ to the CaM N-lobe may induce a conformational change in CT(1520-1669)., (© 2022 Federation of European Biochemical Societies.)

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565-587, called CaM1) decreases the open probability of CNG channels at elevated Ca 2+ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120-1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca 2+ -saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca 2+ -saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447)., (© 2022. The Author(s).)

The Ca 2+ sensor protein, calmodulin (CaM) is ubiquitously expressed in all cells where it binds to hundreds of different target proteins, including dozens of enzymes, receptors, ion channels and numerous Ca 2+ transporters. The only published NMR chemical shift assignments for Ca 2+ -bound CaM (in the absence of a target) have been determined under acidic conditions: at pH 6.5/310 K (BMRB 6541) and pH 6.3/320 K (BMRB 547). However, some CaM/target complexes are not soluble under these conditions. Also, amide chemical shifts are very sensitive to pH and temperature, which can cause large baseline errors when using the existing chemical shift assignments of free CaM to calculate chemical shift perturbations caused by target binding at neutral pH and physiological temperature. We report complete NMR chemical shift assignments of Ca 2+ -saturated CaM under a set of standard conditions at neutral pH and 308 K that will enable more accurate chemical shift comparison between free CaM and CaM/target complexes (BMRB 51289)., (© 2022. The Author(s).)

The neuronal L-type voltage-gated Ca 2+ channel (Ca V 1.2) interacts with Ca 2+ binding protein 1 (CaBP1), that promotes Ca 2+ -induced channel activity. The binding of CaBP1 to the IQ-motif in Ca V 1.2 (residues 1644-1665) blocks the binding of calmodulin and prevents Ca 2+ -dependent inactivation of Ca V 1.2. This Ca 2+ -induced binding of CaBP1 to Ca V 1.2 is important for modulating neuronal synaptic plasticity, which may serve a role in learning and memory. Here we report NMR assignments of the C-terminal domain of CaBP1 (residues 99-167, called CaBP1C) that contains two Ca 2+ bound at the third and fourth EF-hands (EF3 and EF4) and is bound to the Ca V 1.2 IQ-motif from Ca V 1.2 (BMRB accession no. 51518)., (© 2022. The Author(s).)

Retinal cyclic nucleotide-gated (CNG) channels (composed of three CNGA1 and one CNGB1 subunits) exhibit a Ca 2+ -induced reduction in channel open probability mediated by calmodulin (CaM). Defects in the Ca 2+ -dependent regulation of CNG channels may be linked to autosomal recessive retinitis pigmentosa and other inherited forms of blindness. Here, we report the NMR structure and binding analysis of CaM bound to two separate sites within CNGB1 (CaM1: residues 565-589 and CaM2: residues 1120-1147). Our binding studies reveal that CaM1 binds to the Ca 2+ -bound CaM N-lobe with at least fivefold higher affinity than it binds to the CaM C-lobe. By contrast, the CaM2 site binds to the Ca 2+ -bound CaM C-lobe with higher affinity than it binds to the N-lobe. CaM1 and CaM2 both exhibited very weak binding to Ca 2+ -free CaM. We present separate NMR structures of Ca 2+ -saturated CaM bound to CaM1 and CaM2 that define key intermolecular contacts: CaM1 residue F575 interacts with the CaM N-lobe while CaM2 residues L1129, L1132, and L1136 each make close contact with the CaM C-lobe. The CNGB1 mutation F575E abolishes CaM1 binding to the CaM N-lobe while L1132E and L1136E each abolish CaM2 binding to the CaM C-lobe. Thus, a single CaM can bind to both sites in CNGB1 in which the CaM N-lobe binds to CaM1 and the CaM C-lobe binds to CaM2. We propose a Ca 2+ -dependent conformational switch in the CNG channel caused by CaM binding, which may serve to attenuate cGMP binding to CNG channels at high cytosolic Ca 2+ levels in dark-adapted photoreceptors.

Retinal membrane guanylate cyclases (RetGC1 and RetGC2) are expressed in photoreceptor rod and cone cells, where they promote the onset of visual recovery during phototransduction. The catalytic activity of RetGCs is regulated by their binding to regulatory proteins, guanylate cyclase activating proteins (GCAP1-5) and the retinal degeneration 3 protein (RD3). RetGC1 is activated by its binding to Ca 2+ -free/Mg 2+ -bound GCAP1 at low cytosolic Ca 2+ levels in light-activated photoreceptors. By contrast, RetGC1 is inactivated by its binding to Ca 2+ -bound GCAP1 and/or RD3 at elevated Ca 2+ levels in dark-adapted photoreceptors. The Ca 2+ sensitive cyclase activation helps to replenish the cytosolic cGMP levels in photoreceptors during visual recovery. Mutations in RetGC1, GCAP1 or RD3 that disable the Ca 2+ -dependent regulation of cyclase activity are genetically linked to rod/cone dystrophies and other inherited forms of blindness. Here I review the structural interaction of RetGC1 with GCAP1 and RD3. I propose a two-state concerted model in which the dimeric RetGC1 allosterically switches between active and inactive conformational states with distinct quaternary structures that are oppositely stabilized by the binding of GCAP1 and RD3. The binding of Ca 2+ -free/Mg 2+ -bound GCAP1 is proposed to activate the cyclase by stabilizing RetGC1 in an active conformation (R-state), whereas Ca 2+ -bound GCAP1 and/or RD3 inhibit the cyclase by locking RetGC1 in an inactive conformation (T-state). Exposed hydrophobic residues in GCAP1 (residues H19, Y22, M26, F73, V77, W94) are essential for cyclase activation and could be targeted by rational drug design for the possible treatment of rod/cone dystrophies., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ames.)

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679-702) binds tightly to Ca 2+ -bound CaM, which promotes Ca 2+ -induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca 2+ -saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222)., (© 2022. The Author(s).)

Objectives: Chronic diseases account for the majority of healthcare spending. Cirrhosis is a chronic disease whose burden is rising, especially in young adults. This study aimed at describing the direct healthcare costs and utilization in young adults with cirrhosis compared to other chronic diseases common to this age group., Methods: Retrospective population-based study of routinely collected healthcare data from Ontario for the fiscal years 2007-2016 and housed at ICES. Young adults (aged 18-40 years) with cirrhosis, inflammatory bowel disease (IBD), and asthma were identified based on validated case definitions. Total and annual direct healthcare costs and utilization were calculated per individual across multiple healthcare settings and compared based on the type of chronic disease. For cirrhosis, the results were further stratified by etiology and decompensation status., Results: Total direct healthcare spending from 2007 to 2016 increased by 84% for cirrhosis, 50% for IBD, and 41% for asthma. On a per-patient basis, annual costs were the highest for cirrhosis ($6,581/year) compared to IBD ($5,260/year), and asthma ($2,934/year) driven by acute care in cirrhosis and asthma, and drug costs in IBD. Annual costs were four-fold higher in patients with decompensated versus compensated cirrhosis ($20,651/year vs. $5,280/year). Patients with cirrhosis had greater use of both ICU and mental health services., Conclusion: Healthcare costs in young adults with cirrhosis are rising and driven by the use of acute care. Strategies to prevent the development of cirrhosis and to coordinate healthcare in this population through the development of chronic disease prevention and management strategies are urgently needed., Competing Interests: The study sponsors did not participate in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication. No endorsement by ICES or AASLD is intended or should be inferred. Potential conflicts of interest include Dr. Flemming consults for Gilead; Dr. Terrault consults for Exigo, Enyo, PPD, and Entourage and she received grants from Gilead, Genentech, and Roche., (Copyright © 2022 Jeffrey B. Ames et al.)

L-type voltage-gated Ca 2+ channels (CaV1.2 and CaV1.3, called CaV) interact with the Ca 2+ sensor proteins, calmodulin (CaM) and Ca 2+ binding Protein 1 (CaBP1), that oppositely control Ca 2+ -dependent channel activity. CaM and CaBP1 can each bind to the IQ-motif within the C-terminal cytosolic domain of CaV, which promotes increased channel open probability under basal conditions. At elevated cytosolic Ca 2+ levels (caused by CaV channel opening), Ca 2+ -bound CaM binding to CaV is essential for promoting rapid Ca 2+ -dependent channel inactivation (CDI). By contrast, CaV binding to CaBP1 prevents CDI and promotes Ca 2+ -induced channel opening (called CDF). In this review, I provide an overview of the known structures of CaM and CaBP1 and their structural interactions with the IQ-motif to help understand how CaM promotes CDI, whereas CaBP1 prevents CDI and instead promotes CDF. Previous electrophysiology studies suggest that Ca 2+ -free forms of CaM and CaBP1 may pre-associate with CaV under basal conditions. However, previous Ca 2+ binding data suggest that CaM and CaBP1 are both calculated to bind to Ca 2+ with an apparent dissociation constant of ~100 nM when CaM or CaBP1 is bound to the IQ-motif. Since the neuronal basal cytosolic Ca 2+ concentration is ~100 nM, nearly half of the neuronal CaV channels are suggested to be bound to Ca 2+ -bound forms of either CaM or CaBP1 under basal conditions. The pre-association of CaV with calcified forms of CaM or CaBP1 are predicted here to have functional implications. The Ca 2+ -bound form of CaBP1 is proposed to bind to CaV under basal conditions to block CaV binding to CaM, which could explain how CaBP1 might prevent CDI.

Chemical synaptic transmission represents the most sophisticated dynamic process and is highly regulated with optimized neurotransmitter balance. Imbalanced transmitters can lead to transmission impairments, for example, intracellular zinc accumulation is a hallmark of degenerating neurons. However, the underlying mechanisms remain elusive. Postsynaptic density protein-95 (PSD-95) is a primary postsynaptic membrane-associated protein and the major scaffolding component in the excitatory postsynaptic densities, which performs substantial functions in synaptic development and maturation. Its membrane association induced by palmitoylation contributes largely to its regulatory functions at postsynaptic sites. Unlike other structural domains in PSD-95, the N-terminal region (PSD-95NT) is flexible and interacts with various targets, which modulates its palmitoylation of two cysteines (C3/C5) and glutamate receptor distributions in postsynaptic densities. PSD-95NT contains a putative zinc-binding motif (C2H2) with undiscovered functions. This study is the first effort to investigate the interaction between Zn 2+ and PSD-95NT. The NMR titration of 15 N-labeled PSD-95NT by ZnCl 2 was performed and demonstrated Zn 2+ binds to PSD-95NT with a binding affinity (K d ) in the micromolar range. The zinc binding was confirmed by fluorescence and mutagenesis assays, indicating two cysteines and two histidines (H24, H28) are critical residues for the binding. These results suggested the concentration-dependent zinc binding is likely to influence PSD-95 palmitoylation since the binding site overlaps the palmitoylation sites, which was verified by the mimic PSD-95 palmitoyl modification and intact cell palmitoylation assays. This study reveals zinc as a novel modulator for PSD-95 postsynaptic membrane association by chelating its N-terminal region, indicative of its importance in postsynaptic signaling., (© 2021 The Protein Society.)

Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca 2+ and confer Ca 2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg 2+ -bound, Ca 2+ -free, Fe 2+ -free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca 2+ -bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe 2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe 2+ -dependent regulation of cyclase activity in zebrafish photoreceptors.

Postsynaptic density protein-95 (PSD95) contributes to the postsynaptic architecture of neuronal synapses and plays an important role in controlling synaptic plasticity. The N-terminal domain of PSD95 (residues 1-71, called PSD95-NT) interacts with target proteins (calmodulin, α-actinin-1 and CDKL5), which regulate the Ca 2+ -dependent degradation of glutamate receptors. We report complete backbone NMR chemical shift assignments of PSD95-NT (BMRB No. 50752)., (© 2021. The Author(s).)

Retinal guanylate cyclases (RetGCs) promote the Ca 2+ -dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca 2+ -sensitive activation of RetGCs is controlled by a family of photoreceptor Ca 2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg 2+ -bound/Ca 2+ -free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca 2+ levels in light-activated photoreceptors. By contrast, Ca 2+ -bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca 2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca 2+ -dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca 2+ -dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe 2+ and may serve as a Fe 2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.

Case: We describe a case of mild lead poisoning in a 25-year-old woman because of intra-articular migration of lead shot 12 years after gunshot injury to the left hip, ameliorated by arthroscopic foreign body removal. Retained lead can cause systemic symptoms of lead toxicity, supranormal blood lead concentration, and increasingly painful and destructive local arthritis even years after gunshot injury., Conclusion: This report shows that lead fragments should be monitored closely if located near joint spaces. We demonstrate curative therapy for lead poisoning through the use of minimally invasive arthroscopic techniques for removal of retained intra-articular lead missiles., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSCC/B443)., (Copyright © 2021 by The Journal of Bone and Joint Surgery, Incorporated.)

Calmodulin (CaM) regulates the activity of a Ca 2+ channel known as the cardiac ryanodine receptor (RyR2), which facilitates the release of Ca 2+ from the sarcoplasmic reticulum during excitation-contraction coupling in cardiomyocytes. Mutations that disrupt this CaM-dependent channel inactivation result in cardiac arrhythmias. RyR2 contains three different CaM binding sites: CaMBD1 (residues 1940-1965), CaMBD2 (residues 3580-3611), and CaMBD3 (residues 4246-4275). Here, we report a crystal structure of Ca 2+ -bound CaM bound to RyR2 CaMBD3. The structure reveals Ca 2+ bound to the four EF-hands of CaM as well as a fifth Ca 2+ bound to CaM in the interdomain linker region involving Asp81 and Glu85. The CaM mutant E85A abolishes the binding of the fifth Ca 2+ and weakens the binding of CaMBD3 to Ca 2+ -bound CaM. Thus, the binding of the fifth Ca 2+ is important for stabilizing the complex in solution and is not a crystalline artifact. The CaMBD3 peptide in the complex adopts an α-helix (between Phe4246 and Val4271) that interacts with both lobes of CaM. Hydrophobic residues in the CaMBD3 helix (Leu4255 and Leu4259) form intermolecular contacts with the CaM N-lobe, and the CaMBD3 mutations (L4255A and L4259A) each weaken the binding of CaM to RyR2. Aromatic residues on the opposite side of the CaMBD3 helix (Phe4246 and Tyr4250) interact with the CaM C-lobe, but the mutants (F4246A and Y4250A) have no detectable effect on CaM binding in solution. We suggest that the binding of CaM to CaMBD3 and the binding of a fifth Ca 2+ to CaM may contribute to the regulation of RyR2 channel function.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15 Z and 15 E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme . Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward ( 15 Z P r → 15 E P g ) and reverse ( 15 E P g → 15 Z P r ) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-G f intermediate red-shifted from the 15 Z P r photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φ p within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

The αvβ3 integrin, a receptor for many extracellular matrix proteins with RGD-sequence motif, is involved in multiple physiological processes and highly expressed in tumor cells, therefore making it a target for cancer therapy and tumor imaging. Several RGD-containing cyclic octapeptide (named LXW analogs) were screened as αvβ3 antagonists with dramatically different binding affinity, and their structure-activity relationship (SAR) remains elusive. We performed systematic SAR studies and optimized LXW analogs to improve antagonistic potency. The NMR structure of LXW64 was determined and docked to the integrin. Structural comparison and docking studies suggested that the hydrophobicity and aromaticity of the X7 amino acid are highly important for LXW analogs binding to the integrin, a potential hydrophobic pocket on the integrin surface was proposed to play a role in stabilizing the peptide binding. To develop a cost-efficient and fast screening method, computational docking was performed on LXW analogs and compared with in vitro screening. A consistency within the results of both methods was found, leading to the continuous optimization and testing of LXW mutants via in silico screening. Several new LXW analogs were predicted as the integrin antagonists, one of which-LXZ2-was validated by in vitro examination. Our study provides new insight into the RGD recognition specificity and valuable clues for rational design of novel αvβ3 antagonists., Competing Interests: The authors declare no conflict of interest.

In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently discovered protein whose molecular function is unknown. To uncover the molecular mechanism by which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of COA6, which reveals a coiled-coil-helix-coiled-coil-helix domain typical of redox-active proteins found in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the copper-coordinating disulfides of its client proteins, SCO1 and COX2, allowing for copper binding. Finally, our determination of the interaction surfaces and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Calcium-dependent inactivation (CDI) of neuronal voltage-gated Ca 2+ channels (Ca V 1.2) is important for synaptic plasticity, which is associated with learning and memory. The Ca 2+ -dependent binding of calmodulin (CaM) to Ca V 1.2 is essential for CDI. Here we report NMR assignments for a CaM mutant (D21A/D23A/D25A/E32Q/D57A/D59A/N61A/E68Q, called CaM EF12 ) that contains two Ca 2+ bound at the third and fourth EF-hands (EF3 and EF4) and is bound to the IQ-motif (residues 1644-1665) from Ca V 1.2 (BMRB accession no. 27692).

Retinal membrane guanylyl cyclase (RetGC) in photoreceptor rod and cone cells is regulated by a family of guanylyl cyclase activating proteins (GCAP1-7). GCAP5 is expressed in zebrafish photoreceptors and promotes Ca 2+ -dependent regulation of RetGC enzymatic activity that regulates visual phototransduction. We report NMR chemical shift assignments of the Ca 2+ -free activator form of GCAP5 (BMRB No. 27705).

Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in the photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase-activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18-160) that is monomeric and can still bind and negatively regulate RetGC. The NMR solution structure of RD3 revealed an elongated backbone structure (70 Å long and 30 Å wide) consisting of a four-helix bundle with a long unstructured loop between helices 1 and 2. The structure reveals that RD3 residues previously implicated in the RetGC binding map to a localized and contiguous area on the structure, involving a loop between helices 2 and 3 and adjacent parts of helices 3 and 4. The NMR structure of RD3 was validated by mutagenesis. Introducing Trp 85 or Phe 29 to replace Cys or Leu, respectively, disrupts packing in the hydrophobic core and lowers RD3's apparent affinity for RetGC1. Introducing a positive charge at the interface (Glu 32 to Lys) also lowered the affinity. Conversely, introducing Val in place of Cys 93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase. The NMR structure of RD3 presented here provides a structural basis for elucidating RD3-RetGC interactions relevant for normal vision or blindness., (© 2019 Peshenko et al.)

Neuronal calcium sensor (NCS) proteins are EF-hand containing Ca 2+ binding proteins that regulate sensory signal transduction. Many NCS proteins (recoverin, GCAPs, neurocalcin and visinin-like protein 1 (VILIP1)) form functional dimers under physiological conditions. The dimeric NCS proteins have similar amino acid sequences (50% homology) but each bind to and regulate very different physiological targets. Retinal recoverin binds to rhodopsin kinase and promotes Ca 2+ -dependent desensitization of light-excited rhodopsin during visual phototransduction. The guanylyl cyclase activating proteins (GCAP1-5) each bind and activate retinal guanylyl cyclases (RetGCs) in light-adapted photoreceptors. VILIP1 binds to membrane targets that modulate neuronal secretion. Here, I review atomic-level structures of dimeric forms of recoverin, GCAPs and VILIP1. The distinct dimeric structures in each case suggest that NCS dimerization may play a role in modulating specific target recognition. The dimerization of recoverin and VILIP1 is Ca 2+ -dependent and enhances their membrane-targeting Ca 2+ -myristoyl switch function. The dimerization of GCAP1 and GCAP2 facilitate their binding to dimeric RetGCs and may allosterically control the Ca 2+ -dependent activation of RetGCs.

Phytochrome photoreceptors control plant growth, development, and the shade avoidance response that limits crop yield in high-density agricultural plantings. Cyanobacteriochromes (CBCRs) are distantly related photosensory proteins that control cyanobacterial metabolism and behavior in response to light. Photoreceptors in both families reversibly photoconvert between two photostates via photoisomerization of linear tetrapyrrole (bilin) chromophores. Spectroscopic and biochemical studies have demonstrated heterogeneity in both photostates, but the structural basis for such heterogeneity remains unclear. We report solution NMR structures for both photostates of the red/green CBCR NpR6012g4 from Nostoc punctiforme In addition to identifying structural changes accompanying photoconversion, these structures reveal structural heterogeneity for residues Trp655 and Asp657 in the red-absorbing NpR6012g4 dark state, yielding two distinct environments for the phycocyanobilin chromophore. We use site-directed mutagenesis and fluorescence and absorbance spectroscopy to assign an orange-absorbing population in the NpR6012g4 dark state to the minority configuration for Asp657. This population does not undergo full, productive photoconversion, as shown by time-resolved spectroscopy and absorption spectroscopy at cryogenic temperature. Our studies thus elucidate the spectral and photochemical consequences of structural heterogeneity in a member of the phytochrome superfamily, insights that should inform efforts to improve photochemical or fluorescence quantum yields in the phytochrome superfamily., Competing Interests: The authors declare no conflict of interest.

Retinal degeneration 3 protein (RD3) binds to retinal membrane guanylyl cyclase (RetGC) and suppresses the basal activity of RetGC in photoreceptor cells that opposes the allosteric activation of the cyclase by GCAP proteins. Mutations in RD3 that disrupt its inhibition of RetGC are implicated in human retinal degenerative disorders. Here we report both backbone and sidechain NMR assignments for the RD3 protein (BMRB accession no. 27305).

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Retinal guanylyl cyclases (RetGCs) in vertebrate photoreceptors are regulated by the guanylyl cyclase activator proteins (GCAP1 and GCAP2). Here, we report EPR double electron-electron resonance (DEER) studies on the most ubiquitous GCAP isoform, GCAP1 and site-directed mutagenesis analysis to determine an atomic resolution structural model of a GCAP1 dimer. Nitroxide spin-label probes were introduced at individual GCAP1 residues: T29C, E57C, E133C, and E154C. The intermolecular distance of each spin-label probe (measured by DEER) defined restraints for calculating the GCAP1 dimeric structure by molecular docking. The DEER-derived structural model of the GCAP1 dimer was similar within the experimental error for both the Mg2+-bound activator and Ca2+-bound inhibitor states (RMSD < 2.0 Å). The GCAP1 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H19, Y22, F73 and V77. The structural model of the dimer was validated by GCAP1 mutations (H19R, Y22D, F73E, and V77E) at the dimer interface that each abolished protein dimerization. Previous studies have shown that each of these mutants either diminished or completely suppressed the ability of GCAP1 to activate the cyclase. These results suggest that GCAP1 dimerization may affect compartmentalization of GCAP1 in the photoreceptors and/or affect regulation of the cyclase activity.

Objective: To assess diagnostic accuracy and agreement among radiologists in detecting femoroplasty on pre- and post-arthroscopic comparison frog lateral and anteroposterior (AP) pelvic radiographs after treatment of femoroacetabular impingement (FAI) syndrome., Materials and Methods: In this retrospective, cross-sectional study, 86 patients underwent hip arthroscopy (52 with and 34 without femoroplasty) for treatment of FAI syndrome. Three radiologists blinded to clinical data and chronological order of the pre- and post-arthroscopic comparison radiographs independently examined AP pelvis and frog lateral radiographs to detect femoroplasty changes. Statistical analysis outputs included diagnostic accuracy parameters and inter- and intra-observer agreement., Results: Identification of femoroplasty in the frog lateral projection has mean sensitivity 70%, specificity 82%, inter-observer agreement κ 0.74-0.76 and intra-observer agreement κ 0.72-0.85. Using the AP pelvis projection to detect femoroplasty has mean sensitivity 32%, specificity 71%, inter-observer agreement κ 0.47-0.65, and intra-observer agreement κ, 0.56-0.84., Conclusions: Radiologists are only moderately sensitive, though more specific, in femoroplasty detection in the frog lateral projection. The AP pelvis projection yields lower sensitivity and specificity. Both projections have moderate inter- and intra-observer agreement.

Postsynaptic density protein-95 (PSD-95) localizes AMPA-type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca 2+ influx, Ca 2+ /calmodulin (Ca 2+ /CaM) binding to the N-terminus of PSD-95 mediates postsynaptic loss of PSD-95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD-95 N-terminus as important for binding to Ca 2+ /CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaM R 126E , as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca 2+ /CaM to PSD-95 induced by a chronic increase in Ca 2+ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission., (© 2017 The Authors.)

Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two nonconserved cysteine residues (Cys15 and Cys17) that could in principle bind to biologically active transition state metal ions (Zn 2+ and Fe 2+ ). Here, we present nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) binding analyses that demonstrate the binding of one Fe 2+ ion to two GCAP5 molecules (in a 1:2 complex) with a dissociation constant in the nanomolar range. At least one other Fe 2+ binds to GCAP5 with micromolar affinity that likely represents electrostatic Fe 2+ binding to the EF-hand loops. The GCAP5 double mutant (C15A/C17A) lacks nanomolar binding to Fe 2+ , suggesting that Fe 2+ at this site is ligated directly by thiolate groups of Cys15 and Cys17. Size exclusion chromatography analysis indicates that GCAP5 forms a dimer in the Fe 2+ -free and Fe 2+ -bound states. NMR structural analysis and molecular docking studies suggest that a single Fe 2+ ion is chelated by thiol side chains from Cys15 and Cys17 in the GCAP5 dimer, forming an [Fe(SCys) 4 ] complex like that observed previously in two-iron superoxide reductases. Binding of Fe 2+ to GCAP5 weakens its ability to activate photoreceptor human GC-E by decreasing GC activity >10-fold. Our results indicate a strong Fe 2+ -induced inhibition of GC by GCAP5 and suggest that GCAP5 may serve as a redox sensor in visual phototransduction.

Introduction: Orthopedic oncologists often encounter patients with minor bony lesions that are difficult to access surgically and therefore require large exposures out of proportion to the severity of disease that confer significant patient morbidity. Minimally invasive surgical techniques offer the advantage of smaller incisions, shorter operative times, decreased tissue damage, and decreased costs. A variety of surgical procedures have emerged using minimally invasive technologies, particularly in the field of spine surgery. Areas covered: In this article, we describe the Minimal Exposure Tubular Retractor (METRx TM ) System which is a minimally invasive surgical device that utilizes a series of dilators to permit access to a surgical site of interest. This system was developed for use in treatment of disc herniation, spinal stenosis, posterior lumbar interbody fusion, transforaminal lumbar interbody fusion and spinal cord stimulation implantation. We also describe novel uses of this system for minimally invasive biopsy and treatment of benign and metastatic bone lesions at our institution. Expert commentary: Minimally invasive surgical techniques will continue to expand into the field of orthopedic oncology. With a greater number of studies proving the safety and effectiveness of this technique, the demand for minimally invasive treatments will grow.

Estrogen receptor α (ER-α) is a nuclear hormone receptor that controls selected genes, thereby regulating proliferation and differentiation of target tissues, such as breast. Gene expression controlled by ER-α is modulated by Ca 2+ via calmodulin (CaM). Here we present the NMR structure of Ca 2+ -CaM bound to two molecules of ER-α (residues 287-305). The two lobes of CaM bind to the same site on two separate ER-α molecules (residues 292, 296, 299, 302, and 303), which explains why CaM binds two molecules of ER-α in a 1:2 complex and stabilizes ER-α dimerization. Exposed glutamate residues in CaM (Glu-11, Glu-14, Glu-84, and Glu-87) form salt bridges with key lysine residues in ER-α (Lys-299, Lys-302, and Lys-303), which is likely to prevent ubiquitination at these sites and inhibit degradation of ER-α. Transfection of cells with full-length CaM slightly increased the ability of estrogen to enhance transcriptional activation by ER-α of endogenous estrogen-responsive genes. By contrast, expression of either the N- or C-lobe of CaM abrogated estrogen-stimulated transcription of the estrogen responsive genes pS2 and progesterone receptor. These data suggest that CaM-induced dimerization of ER-α is required for estrogen-stimulated transcriptional activation by the receptor. In light of the critical role of ER-α in breast carcinoma, our data suggest that small molecules that selectively disrupt the interaction of ER-α with CaM may be useful in the therapy of breast carcinoma., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity., Competing Interests: The authors have declared that no competing interests exist.

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins with a tetrapyrrole (bilin) chromophore that belong to the phytochrome superfamily. Like phytochromes, CBCRs photoconvert between two photostates with distinct spectral properties. NpR6012g4 from Nostoc punctiforme is a model system for widespread CBCRs with conserved red/green photocycles. Atomic-level structural information for the photoproduct state in this subfamily is not known. Here, we report NMR backbone chemical shift assignments of the light-activated state of NpR6012g4 (BMRB no. 26577) as a first step toward determining its atomic resolution structure.

The regulation and localization of the neuronal voltage gated Ca(2+) channel CaV1.2 is important for synaptic plasticity associated with learning and memory. The cytoskeletal protein, α-actinin-1 is known to interact with CaV1.2 and stabilize its localization at the postsynaptic membrane. Here we report both backbone and sidechain NMR assignments for the C-terminal EF-hands (EF3 and EF4) of α-actinin-1 (residues 824-892, called ACTN&#95;EF34) bound to the IQ-motif (residues 1644-1665) from CaV1.2 (BMRB accession no. 25902).

Cyanobacteriochrome (CBCR) photosensory proteins are phytochrome homologs using bilin chromophores for light sensing across the visible spectrum. NpR6012g4 is a CBCR from Nostoc punctiforme that serves as a model for a widespread CBCR subfamily with red/green photocycles. We report NMR chemical shift assignments for both the protein backbone and side-chain resonances of the red-absorbing dark state of NpR6012g4 (BMRB no. 26582).

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We have previously reported the use of one-bead-one-compound (OBOC) combinatorial technology to develop a disulfide cyclic, Arg-Gly-Asp-containing octapeptide LXW7 (cGRGDdvc), that targets αvβ3 integrin with high affinity and specificity. αvβ3 integrin is known to be overexpressed in many cancers and in tumor vasculature, and it has been established as a cancer therapeutic target. To further optimize LXW7, we have performed systematic structure-activity relationship studies. On the basis of the results, two highly focused OBOC peptide libraries were designed, synthesized, and screened against αvβ3 integrin-transfected K562 cells. One of the best ligands, LXW64, was found to have 6.6-fold higher binding affinity than LXW7, and showed preferential binding to cells expressing αvβ3 integrin. In addition to binding strongly to U-87MG glioblastoma cells in vitro, LXW64 also targets U-87MG xenografts implanted in nude mice, indicating that it is an excellent vehicle for the delivery of cytotoxic payload to tumors and tumor blood vessels that overexpress αvβ3 integrin. Mol Cancer Ther; 15(2); 232-40. ©2015 AACR., (©2015 American Association for Cancer Research.)

The homeobox gene (Hoxd13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of proteins that control embryonic morphogenesis. We report NMR chemical shift assignments of mouse Hoxd13 DNA binding domain bound to an 11-residue DNA duplex (BMRB No. 25133).

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹⁵N-edited ¹H-¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein-chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-Z,anti configuration for the red-absorbing dark state and reveal a C15-E,anti configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4.