Designer molecules of the synaptic organizer MDGA1 reveal 3D conformational control of biological function.

MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors) are synaptic cell surface molecules that regulate the formation of trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs), which promote synaptic development. Mutations in MDGAs are implicated in various neuropsychiatric diseases. MDGAs bind NLGNs in cis on the postsynaptic membrane and physically block NLGNs from binding to NRXNs.

Chemically targeting the redox switch in AP1 transcription factor ΔFOSB.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g.

Interplay between hevin, SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges.

Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges.

Highly Conserved Molecular Features in IgLONs Contrast Their Distinct Structural and Biological Outcomes.

Neuronal growth regulator 1 (NEGR1) and neurotrimin (NTM) are abundant cell-surface proteins found in the brain and form part of the IgLON (Immunoglobulin LSAMP, OBCAM, Neurotrimin) family. In humans, NEGR1 is implicated in obesity and mental disorders, while NTM is linked to intelligence and cognitive function. IgLONs dimerize homophilically and heterophilically, and they are thought to shape synaptic connections and neural circuits by acting in trans (spanning cellular junctions) and/or in cis (at the same side of a junction).

Self-assembly of the bZIP transcription factor ΔFosB.

ΔFosB is a highly stable transcription factor that accumulates in specific brain regions upon chronic exposure to drugs of abuse, stress, or seizures, and mediates lasting behavioral responses. ΔFosB reportedly heterodimerizes with JunD forming a canonical bZIP leucine zipper coiled coil that clamps onto DNA. However, the striking accumulation of ΔFosB protein in brain upon chronic insult has brought its molecular status into question.

Activator Protein-1: redox switch controlling structure and DNA-binding.

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a 'redox switch' centered on an inter-molecular disulfide bond.

Molecular Mechanism of MDGA1: Regulation of Neuroligin 2:Neurexin Trans-synaptic Bridges.

Neuroligins and neurexins promote synapse development and validation by forming trans-synaptic bridges spanning the synaptic cleft. Select pairs promote excitatory and inhibitory synapses, with neuroligin 2 (NLGN2) limited to inhibitory synapses and neuroligin 1 (NLGN1) dominating at excitatory synapses. The cell-surface molecules, MAM domain-containing glycosylphosphatidylinositol anchor 1 (MDGA1) and 2 (MDGA2), regulate trans-synaptic adhesion between neurexins and neuroligins, impacting NLGN2 and NLGN1, respectively.

TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in .

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens.

Discovery of Macrocyclic Pyrimidines as MerTK-Specific Inhibitors.

Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure-based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)-specific inhibitors.

Design and Synthesis of Novel Macrocyclic Mer Tyrosine Kinase Inhibitors.

Mer tyrosine kinase (MerTK) is aberrantly elevated in various tumor cells and has a normal anti-inflammatory role in the innate immune system. Inhibition of MerTK may provide dual effects against these MerTK-expressing tumors through reducing cancer cell survival and redirecting the innate immune response. Recently, we have designed novel and potent macrocyclic pyrrolopyrimidines as MerTK inhibitors using a structure-based approach.

Functional analysis of rare variants found in schizophrenia implicates a critical role for GIT1-PAK3 signaling in neuroplasticity.

Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural circuits via synaptic dysfunction, the underlying molecular mechanisms remain poorly understood. Recent exome sequencing studies of SCZ have uncovered numerous single-nucleotide variants (SNVs); however, the majority of these SNVs have unknown functional consequences, leaving their disease relevance uncertain. Filling this knowledge gap requires systematic application of quantitative and scalable assays to assess known and novel biological functions of genes.

Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme.

Study Hypothesis: Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development.

Study Finding: This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility.

What Is Known Already: Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species.

Computational de novo design of a four-helix bundle protein--DND_4HB.

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence.

Discovery of Mer specific tyrosine kinase inhibitors for the treatment and prevention of thrombosis.

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined.

Pseudo-cyclization through intramolecular hydrogen bond enables discovery of pyridine substituted pyrimidines as new Mer kinase inhibitors.

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors.

Structural determinants of RGS-RhoGEF signaling critical to Entamoeba histolytica pathogenesis.

G protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and guanosine triphosphatase-accelerating protein, EhRGS-RhoGEF, in a nucleotide state-selective fashion.

Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica.

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E.

Biophysical and bioinformatic analyses implicate the Treponema pallidum Tp34 lipoprotein (Tp0971) in transition metal homeostasis.

Metal ion homeostasis is a critical function of many integral and peripheral membrane proteins. The genome of the etiologic agent of syphilis, Treponema pallidum, is compact and devoid of many metabolic enzyme genes. Nevertheless, it harbors genes coding for homologs of several enzymes that typically require either iron or zinc.

Discovery of Novel Small Molecule Mer Kinase Inhibitors for the Treatment of Pediatric Acute Lymphoblastic Leukemia.

Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors.

Increasing sequence diversity with flexible backbone protein design: the complete redesign of a protein hydrophobic core.

Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core.

Computational design of a symmetric homodimer using β-strand assembly.

Computational design of novel protein-protein interfaces is a test of our understanding of protein interactions and has the potential to allow modification of cellular physiology. Methods for designing high-affinity interactions that adopt a predetermined binding mode have proved elusive, suggesting the need for new strategies that simplify the design process. A solvent-exposed backbone on a β-strand is thought of as "sticky" and β-strand pairing stabilizes many naturally occurring protein complexes.

Metal-mediated affinity and orientation specificity in a computationally designed protein homodimer.

Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer.

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation.

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation.

Computational design of the sequence and structure of a protein-binding peptide.

The de novo design of protein-binding peptides is challenging because it requires the identification of both a sequence and a backbone conformation favorable for binding. We used a computational strategy that iterates between structure and sequence optimization to redesign the C-terminal portion of the RGS14 GoLoco motif peptide so that it adopts a new conformation when bound to Gα(i1). An X-ray crystal structure of the redesigned complex closely matches the computational model, with a backbone root-mean-square deviation of 1.

The crystal structure of a self-activating G protein alpha subunit reveals its distinct mechanism of signal initiation.

In animals, heterotrimeric guanine nucleotide-binding protein (G protein) signaling is initiated by G protein-coupled receptors (GPCRs), which activate G protein α subunits; however, the plant Arabidopsis thaliana lacks canonical GPCRs, and its G protein α subunit (AtGPA1) is self-activating. To investigate how AtGPA1 becomes activated, we determined its crystal structure. AtGPA1 is structurally similar to animal G protein α subunits, but our crystallographic and biophysical studies revealed that it had distinct properties.