Association Domain Research Articles

Conformational spread drives the evolution of the calcium–calmodulin protein kinase II

The calcium calmodulin (Ca2+/CaM) dependent protein kinase II (CaMKII) decodes Ca2+ frequency oscillations. The CaMKIIα isoform is predominantly expressed in the brain and has a central role in learning. I matched residue and organismal evolution with collective motions deduced from the atomic structure of the human CaMKIIα holoenzyme to learn how its ring architecture abets function. Protein dynamic simulations showed its peripheral kinase domains (KDs) are conformationally coupled via lateral spread along the central hub. The underlying β-sheet motions in the hub or association domain (AD) were deconvolved into dynamic couplings based on mutual information. They mapped onto a coevolved residue network to partition the AD into two distinct sectors. A second, energetically stressed sector was added to ancient bacterial enzyme dimers for assembly of the ringed hub. The continued evolution of the holoenzyme after AD–KD fusion targeted the sector’s ring contacts coupled to the KD. Among isoforms, the α isoform emerged last and, it alone, mutated rapidly after the poikilotherm–homeotherm jump to match the evolution of memory. The correlation between dynamics and evolution of the CaMKII AD argues single residue substitutions fine-tune hub conformational spread. The fine-tuning could increase CaMKIIα Ca2+ frequency response range for complex learning functions.

Structure of the GAT domain of the endosomal adapter protein Tom1.

Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.

Positive Cooperativity and T286 Autophosphorylation is Observed in a Dimeric Mutant of Calcium-Calmodulin Dependent Protein Kinase II (CaMKII)

CaMKII is a multimeric enzyme that regulates long-term potentiation in the hippocampus. The assembly and organization of 8-14 subunits in a holoenzyme is thought to be required for transduction of calcium spike frequencies, cooperative binding to calmodulin, persistent activation by T286 autophosphorylation, and translocation into synaptic spines. CaMKII assembles into a holoenzyme by virtue of its unique C-terminal association domain (AD). ADs form a central hub-like structure from which regulatory and catalytic domains project. Each AD interacts tightly with three other subunits, two laterally and one transversely, to form a stable core composed of two stacked rings. FRET and analytical centrifugation has indicated that individual catalytic domains can form pairs by virtue of low affinity binding sites. But catalytic domains of the CaMKII mutant lacking AD do not form pairs in physiological conditions. We hypothesize that catalytic domain pairing is the fundamental structure that allows positive cooperativity and T286 autophosphorylation. Furthermore we postulate that high affinity AD core interactions lead to a high local concentration of catalytic domains that allows low-affinity catalytic domain pairing. To test this hypothesis, we mutated the lateral surface of the CaMKIIα AD. When expressed in HEK293 cells, the mutation generated a paired dimeric CaMKIIα. In support of the hypothesis, our study shows that the dimeric enzyme has the same affinity for calmodulin, a similar Hill coefficient for enzymatic activity, and T286 autophosphorylation comparable to that observed in the wild type holoenzyme. In contrast, the monomeric mutant CaMKII lacking AD has lower affinity, no cooperativity, and reduced T286 autophosphorylation. While catalytic domain pairing was observed in the dimeric mutant, biophysical analysis suggests that the nature of the pairing might be altered.

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

Identification of a Novel Tetramerization Domain in Large Conductance K Ca Channels

More than 50 genes are known to encode K + channel monomers and can coassemble to form hetero-tetrameric K + channels. However, only a subset of possible monomer combinations come together to form functional ion channels. The assembly and tetramerization of appropriate channel monomers is mediated by association domains (ADs). To identify such domains in human large-conductance Ca 2+-activated K + channels (hSlo1), we screened hSlo1 domains for self-association using yeast two-hybrid assays. Putative ADs were subjected to functional assays in Xenopus oocytes and further characterized by coprecipitation, native gel electrophoresis, and sucrose density gradient centrifugation assays. This led to the identification of a single intracellular association domain localized near the channel pore and required for channel function. We conclude that this novel tetramerization domain, referred to as BK-T1, promotes the assembly of hSlo1 monomers into functional K Ca channels.

Sequences Surrounding the Src-Homology 3 Domain of Phospholipase Cγ-1 Increase the Domain's Association with Cbl

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCγ1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCγ1 SH3 domain core sequence (aa.796-851) can affect the binding to Cbl, a target used as a prototypical ligand. Consistent with previous reports, our results demonstrated a weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCγ1 SH3 domain but a high-avidity binding to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCγ1 SH3 core domain, however, substantially increased binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCγ1 SH3 domain fusion protein with Cbl was shown to depend entirely upon the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCγ1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCγ1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency.

Preboundary lengthening and intonational and prosodic constituents

An experiment was carried out in order to assess the relation between preboundary lengthening on one hand and intonational and nonintonational prosodic constituents on the other. A number of sentences, realized by an American female speaker, were recorded, manipulated, and subsequently presented to a panel of American listeners. The sentences in each set contained an identical string of segments that occurred before a foot boundary (F), word boundary (W), phonological phrase boundary (P), and an intonational phrase boundary (I). An example is the string [suwprst] in: It's these SUperSTitions that I dislike (F boundary); It's a SUper STAtion they've got there (W boundary); We gave the SUper STAtionary to write with (P boundary); And for the SUper, STEAK will be fine (I boundary). The duration of the preboundary strings was varied in six steps, by lengthening and shortening the original lengths. In addition to the durational makeup of the strings, the intonation contour of the sentences was manipulated. Each of the sentences was resynthesized with two intonation contours: one with rise‐falls on the accented syllables and one in which the initial rise and final fall were bridged. The former contour was assumed to create an intonational boundary between the peaks [the association domain (AD) boundary]. Subjects were asked to rate the preboundary segments as “too long,” “just right,” or “too short.” It was found that the depth of the prosodic boundaries and the presence or absence of AD boundaries require different temporal structuring of the associated preboundary segments. The judgments of the subjects suggest that AD boundaries require longer preboundary segments, whereas the relation between depth of nonintonational boundary and lengthening is less direct. The “durational tolerance” appears to be larger before deeper boundaries. An additional finding was that before AD boundaries significantly more “just‐right” judgments were given, which effect was progressive with prosodic boundary depth.