Evolutionary history expands the range of signaling interactions in hybrid multikinase networks

Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions (‘phylogenetic promiscuity’). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS–GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

Structural characterisation of a novel GPVI nanobody-complex reveals a biologically active domain-swapped GPVI dimer.

GPVI is the major signalling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 (CDR3) loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterised their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nanobodies from six different binding classes showed the strongest binding to recombinant GPVI-Fc suggesting that there was not a single dominant class. The most potent three, Nb2, 21 and 35, inhibited collagen-induced platelet aggregation with nanomolar IC50 values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the CRP binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signalling was abolished in a cell-based NFAT-reporter assay. This demonstrates that the C-C' loop region is important for GPVI signalling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.

Convergent evolution of distinct immune sensor systems for fungal polygalacturonases in Brassicaceae

Plant pattern recognition receptors (PRRs) facilitate recognition of microbial surface patterns and mediate activation of plant immunity. Arabidopsis thaliana RLP42, a leucine-rich repeat (LRR) receptor protein (LRR-RP), senses fungal endopolygalacturonases (PGs) through a ternary complex comprising RLP42, the adapter kinase SOBIR1, and SERK proteins. Several fungal PGs harbor a conserved 9-amino acid fragment pg9(At), which is sufficient to activate RLP42-dependent plant immunity. Domain swap experiments using RLP42 and paralogous RLP40 sequences revealed a dominant role of the island domain (ID) for ligand binding and PRR complex assembly. Involvement of the ID in plant receptor function is reminiscent of plant phytosulfokine (PSK) perception through the receptor, PSKR, a LRR receptor kinase. Sensitivity to pg9(At), which is restricted to A. thaliana, exhibits notable accession specificity as active RLP42 alleles were found in only 16 of 52 accessions tested. Arabidopsis arenosa and Brassica rapa, two Brassicaceae species closely related to A. thaliana, perceive plant immunogenic PG fragments pg20(Aa) or pg36(Bra), which are distinct from pg9(At). Our study unveils unprecedented complexity and dynamics of PG pattern recognition receptor evolution within a single plant family. PG perception systems may have evolved rather independently as a result of convergent evolution even among closely related species.

Structures and an activation mechanism of human potassium-chloride cotransporters

Potassium-chloride cotransporters KCC1 to KCC4 mediate the coupled export of potassium and chloride across the plasma membrane and play important roles in cell volume regulation, auditory system function, and γ-aminobutyric acid (GABA) and glycine-mediated inhibitory neurotransmission. Here, we present 2.9- to 3.6-Å resolution structures of full-length human KCC2, KCC3, and KCC4. All three KCCs adopt a similar overall architecture, a domain-swap dimeric assembly, and an inward-facing conformation. The structural and functional studies reveal that one unexpected N-terminal peptide binds at the cytosolic facing cavity and locks KCC2 and KCC4 at an autoinhibition state. The C-terminal domain (CTD) directly interacts with the N-terminal inhibitory peptide, and the relative motions between the CTD and the transmembrane domain (TMD) suggest that CTD regulates KCCs’ activities by adjusting the autoinhibitory effect. These structures provide the first glimpse of full-length structures of KCCs and an autoinhibition mechanism among the amino acid–polyamine-organocation transporter superfamily.

Exposing the distinctive modular behavior of β-strands and α-helices in folded proteins

Although folded proteins are commonly depicted as simplistic combinations of β-strands and α-helices, the actual properties and functions of these secondary-structure elements in their native contexts are just partly understood. The principal reason is that the behavior of individual β- and α-elements is obscured by the global folding cooperativity. In this study, we have circumvented this problem by designing frustrated variants of the mixed α/β-protein S6, which allow the structural behavior of individual β-strands and α-helices to be targeted selectively by stopped-flow kinetics, X-ray crystallography, and solution-state NMR. Essentially, our approach is based on provoking intramolecular "domain swap." The results show that the α- and β-elements have quite different characteristics: The swaps of β-strands proceed via global unfolding, whereas the α-helices are free to swap locally in the native basin. Moreover, the α-helices tend to hybridize and to promote protein association by gliding over to neighboring molecules. This difference in structural behavior follows directly from hydrogen-bonding restrictions and suggests that the protein secondary structure defines not only tertiary geometry, but also maintains control in function and structural evolution. Finally, our alternative approach to protein folding and native-state dynamics presents a generally applicable strategy for in silico design of protein models that are computationally testable in the microsecond–millisecond regime.

Evolutionarily Driven Domain Swap Alters Sigma Factor Dependence in Bacterial Signaling System

Functional diversity in microbes is driven by vertical or horizontal gene transfer. Using modular bacterial signaling systems, we experimentally validate domain swapping of modular genes. We computationally explore the domain architecture of bacterial two-component systems and find a transcriptional effector domain swap that reconstructed a sigma54-dependent system to become sigma70-dependent, all regulating short-chain carboxylic acid consumption. Verifying their ancestral relationship, we coerce the systems into a pseudo-ancestral state. The differences in regulatory requirements for sigma factors in addition to the characterized system’s involvement in carbon utilization imply that this domain swap reshaped the metabolic potential of proteobacteria. This work provides empirical evidence for a general domain swapping hypothesis, which enables bacteria to shuffle parts of genes to achieve neofunctionalization.

Early and nonredundant functions of dynamin isoforms in clathrin-mediated endocytosis

We use state-of-the-art quantitative total internal reflection fluorescence microscopy to study the isoform-specific functions of dynamin-1 (Dyn1) and dynamin-2 (Dyn2), as well as domain-swap Dyn1/Dyn2 chimeras during early and late stages of clathrin-mediated endocytosis.

The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity and contributes to membrane binding

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.