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HypD and HypC, or its paralogue HybG in Escherichia coli, form the core of the scaffold complex that synthesizes the Fe(CN)2CO component of the bimetallic NiFe‐cofactor of [NiFe]‐hydrogenase. We show here that purified HypC‐HypD and HybG‐HypD complexes catalyse hydrolysis of ATP to ADP (kcat ≅ 0.85·s−1); the ATPase activity of the individual proteins was between 5‐ and 10‐fold lower than that of the complex. Pre‐incubation of HypD with ATP was necessary to restore full activity upon addition of HybG. The conserved Cys41 residue on HypD was essential for full ATPase activity of the complex. Together, our data suggest that HypD undergoes ATP‐dependent conformational activation to facilitate complex assembly in preparation for substrate reduction. The iron–sulfur protein HypD and the small HypC chaperone form the core of the [NiFe]‐hydrogenase maturation machinery. We report here that both proteins have intrinsic ATP‐hydrolyzing activity and that this activity is synergistically enhanced in the HypD‐HypC complex. Our results support a model whereby HypD undergoes ATP‐dependent conformational activation, which allows manifestation of optimal ATPase activity upon HypC binding.

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the -terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and -terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded.

Cancer/testis antigens (CTA) are a heterogeneous group of antigens that are expressed preferentially in tumor cells and testis. Based on this definition the human membrane-associated phospholipase A1 beta (lipase family member I, LIPI) has been identified as CTA. The high homology of LIPI and the membrane-associated phospholipase A1 alpha (lipase family member H, LIPH) suggests that both genes are derived from a common ancestor by gene duplication. In contrast to human LIPI, human LIPH is expressed in several tissues. LIPI sequences have only been identified in mammals. Here, we describe the identification of LIPI in non-mammalian vertebrates. Based on the conserved genomic organization of LIPI and LIPH we identified sequences for both lipases in birds and fishes. In all vertebrates the LIPI locus is neighbored by a member of the RNA binding motif (RBM) family, RBM11. By sequencing of reverse transcriptase-polymerase chain reaction products we determined the sequences of LIPI and LIPH messenger RNA from broilers. We found that the sequence homology between LIPI and LIPH is much higher in non-mammalian species than in mammals. In addition, we found broad expression of LIPI in broilers, resembling the expression profile of LIPH. Our data suggest that LIPI is a CTA only in mammalian species and that the unique sequence features of the mammalian LIPI/RBM11 locus have evolved together with the CTA-like expression pattern of LIPI.

HypD and HypC, or its paralogue HybG in Escherichia coli, form the core of the scaffold complex that synthesizes the Fe(CN)2 CO component of the bimetallic NiFe-cofactor of [NiFe]-hydrogenase. We show here that purified HypC-HypD and HybG-HypD complexes catalyse hydrolysis of ATP to ADP (kcat ≅ 0.85·s-1 ); the ATPase activity of the individual proteins was between 5- and 10-fold lower than that of the complex. Pre-incubation of HypD with ATP was necessary to restore full activity upon addition of HybG. The conserved Cys41 residue on HypD was essential for full ATPase activity of the complex. Together, our data suggest that HypD undergoes ATP-dependent conformational activation to facilitate complex assembly in preparation for substrate reduction.HypD and HypC, or its paralogue HybG in Escherichia coli, form the core of the scaffold complex that synthesizes the Fe(CN)2 CO component of the bimetallic NiFe-cofactor of [NiFe]-hydrogenase. We show here that purified HypC-HypD and HybG-HypD complexes catalyse hydrolysis of ATP to ADP (kcat ≅ 0.85·s-1 ); the ATPase activity of the individual proteins was between 5- and 10-fold lower than that of the complex. Pre-incubation of HypD with ATP was necessary to restore full activity upon addition of HybG. The conserved Cys41 residue on HypD was essential for full ATPase activity of the complex. Together, our data suggest that HypD undergoes ATP-dependent conformational activation to facilitate complex assembly in preparation for substrate reduction.