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DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

Many fundamental cellular processes are controlled via assembly of a network of proteins at membrane surfaces. The proper recruitment of proteins to membranes can be controlled by a wide variety of mechanisms including protein lipidation protein protein interactions posttranslational modifications and binding to specific lipid species present in membranes. There are however only a limited number of analytical techniques that can study the assembly of protein membrane complexes at the molecular level. A relatively new addition to the set of techniques available to study these protein membrane systems is the use of hydrogen deuterium exchange mass spectrometry (HDX MS). HDX MS experiments measure protein conformational dynamics in their native state based on the rate of exchange of amide hydrogens with solvent. This review discusses the use of HDX MS as a tool to identify the interfaces of proteins with membranes and membrane associated proteins as well as define conformational changes elicited by membrane recruitment. Specific examples will focus on the use of HDX MS to examine how large macromolecular protein complexes are recruited and activated on membranes and how both posttranslational modifications and cancer linked oncogenic mutations affect these processes.

Type III phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles and may lead to novel antiviral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design, we have designed novel potent and selective (>1000-fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed antiviral activity against hepatitis C virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as antiviral therapeutics.

Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective.

An analysis of group IVA (GIVA) phospholipase A(2) (PLA(2)) inhibitor binding was conducted using a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models of the GIVA PLA(2) inhibitors pyrrophenone and the 2-oxoamide AX007 docked into the protein were designed on the basis of deuterium exchange results, and extensive molecular dynamics simulations were run to determine protein-inhibitor contacts. The models show that both inhibitors interact with key residues that also exhibit changes in deuterium exchange upon inhibitor binding. Pyrrophenone is bound to the protein through numerous hydrophobic residues located distal from the active site, while the oxoamide is bound mainly through contacts near the active site. We also show differences in protein dynamics around the active site between the two inhibitor-bound complexes. This combination of computational and experimental methods is useful in defining more accurate inhibitor binding sites and can be used in the generation of better inhibitors against GIVA PLA(2).

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) catalyzes release of arachidonic acid from glycerophospholipids, leading to thromboxane A(2) (TxA(2)) production. Some platelet agonists stimulate cPLA(2)alpha, but others require fibrinogen binding to alphaIIbbeta3 to elicit TxA(2). Therefore, relationships between cPLA(2)alpha and alphaIIbbeta3 were examined. cPLA(2)alpha and a cPLA(2)alpha binding partner, vimentin, coimmunoprecipitated with alphaIIbbeta3 from platelets, independent of fibrinogen binding. Studies with purified proteins and with recombinant proteins expressed in CHO cells determined that the interaction between cPLA(2)alpha and alphaIIbbeta3 was indirect and was dependent on the alphaIIb and beta3 cytoplasmic tails. Fibrinogen binding to alphaIIbbeta3 caused an increase in integrin-associated cPLA(2)alpha activity in normal platelets, but not in cPLA(2)alpha-deficient mouse platelets or in human platelets treated with pyrrophenone, a cPLA(2)alpha inhibitor. cPLA(2)alpha activation downstream of alphaIIbbeta3 had functional consequences for platelets in that it was required for fibrinogen-dependent recruitment of activated protein kinase Cbeta to the alphaIIbbeta3 complex and for platelet spreading. Thus, cPLA(2)alpha and alphaIIbbeta3 interact to reinforce each other's functions during alphaIIbbeta3 signaling. This provides a plausible explanation for the role of alphaIIbbeta3 in TxA(2) formation and in the defective hemostatic function of mouse or human platelets deficient in cPLA(2)alpha.