Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

Abstract Abstract 774 Introduction: The platelet Glycoprotein Ib/V/IX (GpIb/V/IX) complex is considered a major target for anticoagulant therapy. The primary function of the receptor is to mediate platelet adhesion to von Willebrand factor (VWF) bound to damaged sub-endothelium. This represents the first critical step for platelet adhesion under conditions of high fluid shear stress. GpIb/V/IX is implicated in a number of thrombotic pathological processes such as stroke or myocardial infarction and the bleeding disorders Bernard-Soulier syndrome, platelet type von Willebrand disease (Pt-VWD) and thrombotic thrombocytopenic purpura. We have successfully determined the structure of the GpIbalpha N-terminal domain in complex with a potent (sub nM) 11meric peptide inhibitor (OS1) of the interaction with VWF. Methods: We have determined the crystal structure to 1.8Å resolution using molecular replacement. Results. The peptide sequence CTERMALHNLC was readily identifiable bound to GpIbalpha between the extended regulatory (R) loop and the concave surface of the leucine rich repeats. The peptide adopts one and a half turns of an alpha-helix and contacts three subsites (S1, S2 and S3). S1 and S2 reside within the leucine rich repeats and S3 has a unique feature as this subsite involves contact with the regulatory R-loop stabilizing it in a well defined conformation with helical character. This loop alters conformation between an extended beta-hairpin in the VWF-A1 bound structure and a more compact largely disordered structure in the unliganded structure. In this regard, the Pt-VWD mutations of GpIbalpha, G233V and M239V, which reside in the R-loop act by inducing a beta-conformation and thus result in a high affinity form of the receptor. Conclusions: These studies provide a strategy for targeting the GpIbalpha-VWF interaction using small molecules or alpha-helical peptides exploiting the GpIbalpha subsites described here and acting allosterically to stabilise a low affinity conformation of the receptor with an alpha helical R-loop. Ligand mimetic peptide complex crystal structures for the platelet receptors integrin aIIbb3 with RGD, and alpha2beta1 with a collagen peptide have been described and the former are currently in therapeutic use for treatment of thromboemboletic disorders. Targeting the GpIbalpha-VWF interaction may provide anti-thrombotic drugs which affect platelet adhesion under high shear stress without compromising normal processes of platelet adhesion and aggregation which may be required for normal hemostasis to function. Targeting protein-protein interactions is considered one of the great contemporary challenges in drug discovery. The understanding of how the S1S2S3 subsites provide very effective inhibition of a large protein-protein interaction has wide applicability. LRR proteins are an extended family mediating protein-protein interactions involved in a variety of disease processes such as sepsis, asthma, immunodeficiencies, atherosclerosis, alzheimers (leucine rich repeat kinase) and leukaemia (leucine rich repeat phosphatase). The structural fit of the helical curvature of the peptide with the arc of the leucine rich repeats may provide a basis for further development of alpha-helical peptide mimetics targeting other members of the LRR family which utilize the concave face. Disclosures: No relevant conflicts of interest to declare.

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Ribosome-binding protein p34 is a member of the leucine-rich-repeat-protein superfamily

Biochemical Journal ◽

10.1042/bj2940465 ◽

1993 ◽

Vol 294 (2) ◽

pp. 465-472 ◽

Cited By ~ 19

Author(s):

T Ohsumi ◽

T Ichimura ◽

H Sugano ◽

S Omata ◽

T Isobe ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Tandem Repeats ◽

Structural Similarity ◽

Cytoplasmic Domain ◽

Structural Features ◽

Binding Activity ◽

Leucine Rich Repeat ◽

Ribosome Binding

Protein p34 is a non-glycosylated membrane protein characteristic of rough microsomes and is believed to play a role in the ribosome-membrane association. In the present study we isolated cDNA encoding p34 from a rat liver cDNA library and determined its complete amino acid sequence. p34 mRNA is 3.2 kb long and encodes a polypeptide of 307 amino acids with a molecular mass of about 34.9 kDa. Primary sequence analysis, coupled with biochemical studies on the topology, suggested that p34 is a type II signal-anchor protein; it is composed of a large cytoplasmic domain, a membrane-spanning segment and a 38-amino-acid-long luminally disposed C-terminus. The cytoplasmic domain of p34 has several noteworthy structural features, including a region of 4.5 tandem repeats of 23-24 amino acids. The repeated motif shows structural similarity to the leucine-rich repeat which is found in a variety of proteins widely distributed among eukaryotic cells and which potentially functions in mediating protein-protein interactions. The cytoplasmic domain also contains a characteristic hydrophilic region with abundant charged amino acids. These structural regions may be important for the observed ribosome-binding activity of the p34 protein.

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A Leucine Rich Repeat Protein Provides a SHOC2 the RAS Circuit: A Structure-Function Perspective

Molecular and Cellular Biology ◽

10.1128/mcb.00627-20 ◽

2021 ◽

Author(s):

Jason J. Kwon ◽

William C. Hahn

Keyword(s):

Protein Interactions ◽

Structural Features ◽

Mitogen Activated Protein Kinase ◽

Ras Signaling ◽

Leucine Rich Repeat ◽

Repeat Protein ◽

Adaptive Resistance ◽

Binding Partners ◽

Mapk Inhibitors ◽

Leucine Rich Repeat Protein

SHOC2 is a prototypical leucine-rich repeat protein that promotes downstream receptor tyrosine kinase (RTK)/RAS signaling and plays important roles in several cellular and developmental processes. Gain-of-function germline mutations of SHOC2 drive the RASopathy, Noonan-like syndrome, and SHOC2 mediates adaptive resistance to mitogen-activated protein kinase (MAPK) inhibitors. Similar to many scaffolding proteins, SHOC2 facilitates signal transduction by enabling proximal protein interactions and regulating the subcellular localization of its binding partners. Here we review the structural features of SHOC2 that mediate its known functions, discuss these elements in the context of various binding partners and signaling pathways, and highlight areas of SHOC2 biology where a consensus view has not yet emerged.

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Structural characterization of Treponema pallidum Tp0225 reveals an unexpected leucine-rich repeat architecture

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19007726 ◽

2019 ◽

Vol 75 (7) ◽

pp. 489-495

Author(s):

Raghavendran Ramaswamy ◽

Simon Houston ◽

Bianca Loveless ◽

Caroline E. Cameron ◽

Martin J. Boulanger

Keyword(s):

Protein Interactions ◽

Structural Characterization ◽

Pathogenic Bacteria ◽

Treponema Pallidum ◽

Host Cells ◽

Leucine Rich Repeat ◽

Solvent Molecules ◽

Molecular Landscape ◽

Lrr Domain

The phylogenetically divergent spirochete bacterium Treponema pallidum subsp. pallidum is the causative agent of syphilis. Central to the capacity of T. pallidum to establish infection is the ability of the pathogen to attach to a diversity of host cells. Many pathogenic bacteria employ leucine-rich repeat (LRR) domain-containing proteins to mediate protein–protein interactions, including attachment to host components and establishment of infection. Intriguingly, T. pallidum expresses only one putative LRR domain-containing protein (Tp0225) with an unknown function. In an effort to ascribe a function to Tp0225, a comprehensive phylogenetic analysis was first performed; this investigation revealed that Tp0225 clusters with the pathogenic clade of treponemes. Its crystal structure was then determined to 2.0 Å resolution using Pt SAD phasing, which revealed a noncanonical architecture containing a hexameric LRR core with a discontinuous β-sheet bridged by solvent molecules. Furthermore, a surface-exposed, hydrophobic pocket, which was found in Tp0225 but is largely absent in canonical LRR domains from other pathogenic bacteria, may serve to coordinate a hydrophobic ligand. Overall, this study provides the first structural characterization of the sole LRR domain-containing protein from T. pallidum and offers insight into the unique molecular landscape of this important human pathogen.

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How Parkinson's disease-related mutations disrupt the dimerization of WD40 domain in LRRK2: a comparative molecular dynamics simulation study

Physical Chemistry Chemical Physics ◽

10.1039/d0cp03171b ◽

2020 ◽

Vol 22 (36) ◽

pp. 20421-20433

Author(s):

Xinyi Li ◽

Mingyu Ye ◽

Yue Wang ◽

Ming Qiu ◽

Tingting Fu ◽

...

Keyword(s):

Parkinson’S Disease ◽

Molecular Dynamics ◽

Parkinson's Disease ◽

Molecular Dynamics Simulation ◽

Neurodegenerative Disease ◽

Simulation Study ◽

Dynamics Simulation ◽

Pathogenic Factor ◽

Leucine Rich Repeat ◽

Wd40 Domain

The multidomain kinase enzyme leucine-rich-repeat kinase 2 (LRRK2), activated through a homodimerization manner, is identified as an important pathogenic factor in Parkinson's disease (PD), the second most common neurodegenerative disease wordwide.

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The CCR4 protein from Saccharomyces cerevisiae contains a leucine-rich repeat region which is required for its control of ADH2 gene expression.

Genetics ◽

10.1093/genetics/132.4.951 ◽

1992 ◽

Vol 132 (4) ◽

pp. 951-962 ◽

Cited By ~ 6

Author(s):

T Malvar ◽

R W Biron ◽

D B Kaback ◽

C L Denis

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Interactions ◽

Growth Conditions ◽

Temperature Sensitive ◽

Leucine Rich Repeat ◽

Repeat Region ◽

Protein Protein Interactions ◽

Reading Frame ◽

Cloned Gene ◽

Chromosome I

Abstract The CCR4 gene from Saccharomyces cerevisiae is required for the transcription of the glucose-repressible alcohol dehydrogenase (ADH2). Mutations in CCR4 also suppress the transcription at the ADH2 and his4-912delta loci caused by defects in the SPT10 (CRE1) and SPT6 (CRE2) genes. The CCR4 gene was mapped to the left arm of chromosome I and cloned by complementation of function using previously isolated segments of chromosome I. DNA sequence analysis of the cloned gene defined CCR4 as a 2511 bp open reading frame that would encode a polypeptide of 837 amino acids. The CCR4 mRNA was found to be 2.8 kb in size and Western analysis identified CCR4 as a 95,000 D protein. Disruption of the CCR4 gene resulted in reduced levels of ADH2 expression under both glucose and ethanol growth conditions and in temperature sensitive growth on nonfermentative medium, phenotypes essentially indistinguishable from previously identified mutations in CCR4. The amino terminus of the CCR4 protein was found to be rich in glutamine residues similar to a number of genes which are required for transcription. More importantly, CCR4 showed similarity to a diverse set of proteins sharing a leucine-rich tandem repeat motif, the presence of which has been implicated in mediating protein-protein interactions. Deletions of several of the five leucine-rich repeats in CCR4 were shown to produce nonfunctional proteins indicating the importance of the repeats to CCR4 activity. This leucine-rich repeat region may mediate the contact CCR4 makes with another factor.

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LRX Proteins play a crucial role in pollen grain and pollen tube cell wall development

10.1101/223008 ◽

2017 ◽

Cited By ~ 2

Author(s):

Tohnyui Ndinyanka Fabrice ◽

Hannes Vogler ◽

Christian Draeger ◽

Gautam Munglani ◽

Shibu Gupta ◽

...

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Pollen Tube ◽

Protein Interactions ◽

Molecular Techniques ◽

Fine Tuning ◽

Cell Wall Formation ◽

Leucine Rich Repeat ◽

Biophysical Parameters ◽

Wall Formation

AbstractLeucine-rich repeat extensins (LRXs) are chimeric proteins containing an N-terminal leucine-rich repeat (LRR) and a C-terminal extensin domain. LRXs are involved in cell wall formation in vegetative tissues and required for plant growth. However, the nature of their role in these cellular processes remains to be elucidated. Here, we used a combination of molecular techniques, light microscopy, and transmission electron microscopy to characterize mutants of pollen-expressed LRXs in Arabidopsis thaliana. Mutations in multiple pollen-expressed lrx genes causes severe defects in pollen germination and pollen tube (PT) growth, resulting in a reduced seed set. Physiological experiments demonstrate that manipulating Ca2+ availability partially suppresses the PT growth defects, suggesting that LRX proteins influence Ca2+-related processes. Furthermore, we show that LRX protein localizes to the cell wall, and its LRR-domain (which likely mediates protein-protein interactions) is associated with the plasma membrane. Mechanical analyses by cellular force microscopy and finite element method-based modelling revealed significant changes in the material properties of the cell wall and the fine-tuning of cellular biophysical parameters in the mutants compared to the wild type. The results indicate that LRX proteins might play a role in cell wall-plasma membrane communication, influencing cell wall formation and cellular mechanics.

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High-resolution crystal structure of the leucine-rich repeat domain of the human tumour suppressor PP32A (ANP32A)

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15006457 ◽

2015 ◽

Vol 71 (6) ◽

pp. 684-687 ◽

Cited By ~ 3

Author(s):

Sara Zamora-Caballero ◽

Lina Šiaučiunaite-Gaubard ◽

Jeronimo Bravo

Keyword(s):

High Resolution ◽

Protein Interactions ◽

Crystal Packing ◽

Tumour Suppressor ◽

Small Displacement ◽

Leucine Rich Repeat ◽

Leucine Rich Repeat Domain ◽

Repeat Domain ◽

Α Helix ◽

Complex Crystals

Acidic leucine-rich nuclear phosphoprotein 32A (PP32A) is a tumour suppressor whose expression is altered in many cancers. It is an apoptotic enhancer that stimulates apoptosome-mediated caspase activation and also forms part of a complex involved in caspase-independent apoptosis (the SET complex). Crystals of a fragment of human PP32A corresponding to the leucine-rich repeat domain, a widespread motif suitable for protein–protein interactions, have been obtained. The structure has been refined to 1.56 Å resolution. This domain was previously solved at 2.4 and 2.69 Å resolution (PDB entries 2je0 and 2je1, respectively). The new high-resolution structure shows some differences from previous models: there is a small displacement in the turn connecting the first α-helix (α1) to the first β-strand (β1), which slightly changes the position of α1 in the structure. The shift in the turn is observed in the context of a new crystal packing unrelated to those of previous structures.

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Identification, biochemical characterization and crystallization of the central region of human ATG16L1

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17013280 ◽

2017 ◽

Vol 73 (10) ◽

pp. 560-567 ◽

Cited By ~ 2

Author(s):

Archna Archna ◽

Andrea Scrima

Keyword(s):

Protein Interactions ◽

Biochemical Characterization ◽

Structural Information ◽

Full Length ◽

Protein Protein Interactions ◽

Autophagosome Formation ◽

Molecular Scaffold ◽

Crystal Forms ◽

Wd40 Domain ◽

High Instability

ATG16L1 plays a major role in autophagy. It acts as a molecular scaffold which mediates protein–protein interactions essential for autophagosome formation. The ATG12~ATG5–ATG16L1 complex is one of the key complexes involved in autophagosome formation. Human ATG16L1 comprises 607 amino acids with three functional domains named ATG5BD, CCD and WD40, where the C-terminal WD40 domain represents approximately 50% of the full-length protein. Previously, structures of the C-terminal WD40 domain of human ATG16L1 as well as of human ATG12~ATG5 in complex with the ATG5BD of ATG16L1 have been reported. However, apart from the ATG5BD, no structural information for the N-terminal half, including the CCD, of human ATG16L1 is available. In this study, the authors aimed to structurally characterize the N-terminal half of ATG16L1. ATG16L111–307 in complex with ATG5 has been purified and crystallized in two crystal forms. However, both crystal structures revealed degradation of ATG16L1, resulting in crystals comprising only full-length ATG5 and the ATG5BD of ATG16L1. The structures of ATG5–ATG5BD in two novel crystal forms are presented, further supporting the previously observed dimerization of ATG5–ATG16L1. The reported degradation points towards a high instability at the linker region between the ATG5BD and the CCD in ATG16L1. Based on this observation and further biochemical analysis of ATG16L1, a stable 236-amino-acid subfragment comprising residues 72–307 of the N-terminal half of ATG16L1, covering the residual, so far structurally uncharacterized region of human ATG16L1, was identified. Here, the identification, purification, biochemical characterization and crystallization of the proteolytically stable ATG16L172–307 subfragment are reported.

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