Coiled Coils - A Model System for the 21st Century.

α-Helical coiled coils were described more than 60 years ago as simple, repetitive structures mediating oligomerization and mechanical stability. Over the past 20 years, however, they have emerged as one of the most diverse protein folds in nature, enabling many biological functions beyond mechanical rigidity, such as membrane fusion, signal transduction, and solute transport. Despite this great diversity, their structures can be described by parametric equations, making them uniquely suited for rational protein design. Far from having been exhausted as a source of structural insight and a basis for functional engineering, coiled coils are poised to become even more important for protein science in the coming decades.

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q0 or Q0 H2 ). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q0 and O2 , or NADH and Q0 H2 respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.

Structural diversity of coiled coils in protein fibers of the bacterial cell envelope.

The cell envelope of bacteria shows great diversity in architecture and composition, to a large extent due to its proteome. Proteins localized to the cell envelope, whether integrally embedded in the membrane, membrane-anchored, or peripherally associated as part of a macromolecular complex, often form elongated fibers, in which coiled coils represent a prominent structural element. These coiled-coil segments show a surprising degree of structural variability, despite being shaped by a small number of simple biophysical rules, foremost being their geometry of interaction referred to as 'knobs-into-holes'. Here we will review this diversity, particularly as it has emerged over the last decade.

The Structure and Topology of α-Helical Coiled Coils.

α-Helical coiled coils constitute one of the most diverse folds yet described. They range in length over two orders of magnitude; they form rods, segmented ropes, barrels, funnels, sheets, spirals, and rings, which encompass anywhere from two to more than 20 helices in parallel or antiparallel orientation; they assume different helix crossing angles, degrees of supercoiling, and packing geometries. This structural diversity supports a wide range of biological functions, allowing them to form mechanically rigid structures, provide levers for molecular motors, project domains across large distances, mediate oligomerization, transduce conformational changes and facilitate the transport of other molecules. Unlike almost any other protein fold known to us, their structure can be computed from parametric equations, making them an ideal model system for rational protein design. Here we outline the principles by which coiled coils are structured, review the determinants of their folding and stability, and present an overview of their diverse architectures.

A domain dictionary of trimeric autotransporter adhesins.

Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive outer membrane proteins that mediate adhesion to external surfaces in many Gram-negative bacteria. In recent years, several TAAs have been investigated in considerable detail, also at the structural level. However, in their vast majority, putative TAAs in prokaryotic genomes remain poorly annotated, due to their sequence diversity and changeable domain architecture. In order to achieve an automated annotation of these proteins that is both detailed and accurate we have taken a domain dictionary approach, in which we identify recurrent domains by sequence comparisons, produce bioinformatic descriptors for each domain type, and connect these to structural information where available. We implemented this approach in a web-based platform, daTAA, in 2008 and demonstrated its applicability by reconstructing the complete fiber structure of a TAA conserved in enterobacteria. Here we review current knowledge on the domain structure of TAAs.

Adenylate cyclases: Receivers, transducers, and generators of signals.

Class III adenylate cyclases (ACs) are widespread signaling proteins, which translate diverse intracellular and extracellular stimuli into a uniform intracellular signal. They are typically composed of an N-terminal array of input domains and transducers, followed C-terminally by a catalytic domain, which, as a dimer, generates the second messenger cAMP. The input domains, which receive stimuli, and the transducers, which propagate the signals, are often found in other signaling proteins. The nature of stimuli and the regulatory mechanisms of ACs have been studied experimentally in only a few cases, and even in these, important questions remain open, such as whether eukaryotic ACs regulated by G protein-coupled receptors can also receive stimuli through their own membrane domains. Here we survey the current knowledge on regulation and intramolecular signal propagation in ACs and draw comparisons to other signaling proteins. We highlight the pivotal role of a recently identified cyclase-specific transducer element located N-terminally of many AC catalytic domains, suggesting an intramolecular signaling capacity.

Characterization of a novel signal transducer element intrinsic to class IIIa/b adenylate cyclases and guanylate cyclases.

Adenylate cyclases (ACs) are signaling proteins that produce the second messenger cAMP. Class III ACs comprise four groups (class IIIa-d) of which class IIIa and IIIb ACs have been identified in bacteria and eukaryotes. Many class IIIa ACs are anchored to membranes via hexahelical domains. In eukaryotic ACs, membrane anchors are well conserved, suggesting that this region possesses important functional characteristics that are as yet unknown. To address this question, we replaced the hexahelical membrane anchor of the mycobacterial AC Rv1625c with the hexahelical quorum-sensing receptor from Legionella, LqsS. Using this chimera, we identified a novel 19-amino-acid cyclase transducer element (CTE) located N-terminally to the catalytic domain that links receptor stimulation to effector activation. Coupling of the receptor to the AC was possible at several positions distal to the membrane exit, resulting in stimulatory or inhibitory responses to the ligand Legionella autoinducer-1. In contrast, on the AC effector side functional coupling was only successful when starting with the CTE. Bioinformatics approaches established that distinct CTEs are widely present in class IIIa and IIIb ACs and in vertebrate guanylate cyclases. The data suggest that membrane-delimited receiver domains transduce regulatory signals to the downstream catalytic domains in an engineered AC model system. This may suggest a previously unknown mechanism for cellular cAMP regulation.

Regulation by the quorum sensor from Vibrio indicates a receptor function for the membrane anchors of adenylate cyclases.

Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 µM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates.

α/ß coiled coils.

Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short ß-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/ß coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold.