Structure of dystrophia myotonica protein kinase

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled‐coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM‐8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled‐coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully‐ordered and correctly positioned αC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C‐terminal extension of the kinase domain is bound to the N‐terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C‐terminal extension compared to the closely related Rho‐associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM‐8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.     

Nucleotide recognition by the initiation factor aIF5B: Free energy simulations of a neoclassical GTPase

The GTPase aIF5B is a universally conserved initiation factor that assists ribosome assembly. Crystal structures of its nucleotide complexes, X‐ray(GTP) and X‐ray(GDP), are similar in the nucleotide vicinity, but differ in the orientation of a distant domain IV. This has led to two, contradictory, mechanistic models. One postulates that X‐ray(GTP) and X‐ray(GDP) are, respectively, the active, “ON” and the inactive, “OFF” states; the other postulates that both structures are OFF, whereas the ON state is still uncharacterized. We study GTP/GDP binding using molecular dynamics and a continuum electrostatic free energy method. We predict that X‐ray(GTP) has a ≈ 3 kcal/mol preference to bind GDP, apparently contradicting its assignment as ON. However, the preference arises mainly from a single, nearby residue from the switch 2 motif: Glu81, which becomes protonated upon GTP binding, with a free energy cost of about 4 kcal/mol. We then propose a different model, where Glu81 protonation/deprotonation defines the ON/OFF states. With this model, the X‐ray(GTP):GTP complex, with its protonated Glu81, is ON, whereas X‐ray(GTP):GDP is OFF. The model postulates that distant conformational changes such as domain IV rotation are “uncoupled” from GTP/GDP exchange and do not affect the relative GTP/GDP binding affinities. We analyze the model using a general thermodynamic framework for GTPases. It yields rather precise predictions for the nucleotide specificities of each state, and the state specificities of each nucleotide, which are roughly comparable to the homologues IF2 and aIF2, despite the lack of any conformational switching in the model. © 2012 Wiley Periodicals, Inc.     

Partially folded structure of flavin adenine dinucleotide-depleted ferredoxin-NADP+ reductase with residual NADP+ binding domain

Maize ferredoxin-NADP(+) reductase (FNR) consists of flavin adenine dinucleotide (FAD) and NADP(+) binding domains with a FAD molecule bound noncovalently in the cleft between these domains. The structural changes of FNR induced by dissociation of FAD have been characterized by a combination of optical and biochemical methods. The CD spectrum of the FAD-depleted FNR (apo-FNR) suggested that removal of FAD from holo-FNR produced an intermediate conformational state with partially disrupted secondary and tertiary structures. Small angle x-ray scattering indicated that apo-FNR assumes a conformation that is less globular in comparison with holo-FNR but is not completely chain-like. Interestingly, the replacement of tyrosine 95 responsible for FAD binding with alanine resulted in a molecular form similar to apo-protein of the wild-type enzyme. Both apo- and Y95A-FNR species bound to Cibacron Blue affinity resin, indicating the presence of a native-like conformation for the NADP(+) binding domain. On the other hand, no evidence was found for the existence of folded conformations in the FAD binding domains of these proteins. These results suggested that FAD-depleted FNR assumes a partially folded structure with a residual NADP(+) binding domain but a disordered FAD binding domain.     

Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag-like K(+)) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization-activated cyclic nucleotide-gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the "self-liganded" conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating.