Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with respect to membrane protein structure prediction and membrane protein stability.

Activation of the visual pigment rhodopsin is caused by 11-cis to -trans isomerization of its retinal chromophore. High-resolution solid-state NMR measurements on both rhodopsin and the metarhodopsin II intermediate show how retinal isomerization disrupts helix interactions that lock the receptor off in the dark. We made 2D dipolar-assisted rotational resonance NMR measurements between 13 C-labels on the retinal chromophore and specific 13 C-labels on tyrosine, glycine, serine, and threonine in the retinal binding site of rhodopsin. The essential aspects of the isomerization trajectory are a large rotation of the C20 methyl group toward extracellular loop 2 and a 4-to 5-Å translation of the retinal chromophore toward transmembrane helix 5. The retinal-protein contacts observed in the active metarhodopsin II intermediate suggest a general activation mechanism for class A G proteincoupled receptors involving coupled motion of transmembrane helices 5, 6, and 7.G protein-coupled receptors (GPCRs) are a large superfamily of membrane receptors that have seven transmembrane (TM) helices and respond to a wide array of signaling ligands. Similarities in the sequences of these receptors have led to the idea that they share a common activation mechanism, whereas sequence diversity is correlated with the specificity of different receptors for different ligands and G proteins. With the exception of the visual pigment rhodopsin (1, 2), the structures of GPCRs are unknown. The rhodopsin crystal structure shows that the TM helices are locked in an inactive conformation by interhelical interactions involving conserved amino acids. The general model of GPCR activation that has emerged over the past few years is that ligand-binding, or retinal isomerization in the case of the visual pigments, disrupts these interactions and drives a rigid body movement of one or more of the TM helices (3, 4).Helix-helix interactions in rhodopsin are mediated by two sets of conserved amino acids. The highly conserved signature amino acids have sequence identities of Ͼ80% across the family of class A GPCRs. These amino acids are generally highly polar, aromatics, or proline. The group-conserved amino acids are small and͞or weakly polar (Gly, Ala, Ser, Thr, and Cys) (5). They have low individual sequence identities but are highly conserved (Ͼ80%) when considered as a group. These amino acids are involved in mediating close helix contacts and facilitating interhelical hydrogen bonding (6).The location of the group-conserved amino acids largely in the interfaces of TM helices H1-H4 of rhodopsin has suggested that these helices are locked in a stable structure that does not change significantly upon receptor activation (5). There are fewer group-conserved amino acids in H5-H7. H5 and H7 are unusual in containing conserved prolines that facilitate interactions with the H1-H4 core by exposing the i-4 backbone carbonyl for interhelical hydrogen bonding. H6 is unusual in that the TM region is composed of conserved aromatic and large hydrophobic amino acids ...

Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACβ) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.

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Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.