1.35 and 2.07 Å resolution structures of the red abalone sperm lysin monomer and dimer reveal features involved in receptor binding

Abalone sperm use lysin to make a hole in the egg's protective vitelline envelope (VE). When released from sperm, lysin first binds to the VE receptor for lysin (VERL) then dissolves the VE by a non-enzymatic mechanism. The structures of the monomeric and dimeric forms of Haliotis rufescens (red abalone) lysin, previously solved at 1.90 and 2.75 Å, respectively, have now been refined to 1.35 and 2.07 Å, respectively. The monomeric form of lysin was refined using previously obtained crystallization conditions, while the dimer was solved in a new crystal form with four molecules (two dimers) per asymmetric unit. These high-resolution structures reveal alternate residue conformations, enabling a thorough analysis of the conserved residues contributing to the amphipathic nature of lysin. The availability of five independent high-resolution copies of lysin permits comparisons leading to insights on the local flexibility of lysin and alternative conformations of the hypervariable N-terminus, thought to be involved in species-specific receptor recognition. The new analysis led to the discovery of the basic nature of a cleft formed upon dimerization and a patch of basic residues in the dimer interface. Identification of these features was not possible at lower resolution. In light of this new information, a model explaining the binding of sperm lysin to egg VERL and the subsequent dissolution of the egg VE is proposed.

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Solution structure of sperm lysin yields novel insights into molecular dynamics of rapid protein evolution

10.1101/160580 ◽

2017 ◽

Cited By ~ 1

Author(s):

Damien B. Wilburn ◽

Lisa M. Tuttle ◽

Rachel E. Klevit ◽

Willie J. Swanson

Keyword(s):

Protein Evolution ◽

Solution Structure ◽

Biochemical Characterization ◽

Arms Race ◽

Targeted Mutagenesis ◽

Marine Mollusk ◽

Haliotis Rufescens ◽

Binding Interface ◽

Red Abalone ◽

Species Specific

AbstractProtein evolution is driven by the sum of different physiochemical and genetic processes that usually results in strong purifying selection to maintain biochemical functions. However, proteins that are part of systems under arms race dynamics often evolve at unparalleled rates that can produce atypical biochemical properties. In the marine mollusk abalone, lysin and VERL are a pair of rapidly coevolving proteins that are essential for species-specific interactions between sperm and egg. Despite extensive biochemical characterization of lysin, including crystal structures of multiple orthologs, it was unclear how sites under positive selection may facilitate recognition of VERL. Using a combination of targeted mutagenesis and multidimensional NMR, we present a high-definition solution structure of sperm lysin from red abalone (Haliotis rufescens). Unapparent from the crystallography data, multiple NMR-based analyses conducted in solution reveal clustering of the N-and C-termini to form a nexus of 13 positively selected sites that constitute a VERL binding interface. Evolutionary rate was found to be a significant predictor of backbone flexibility, which may be critical for lysin bioactivity and / or accelerated evolution. These flexible, rapidly evolving segments that constitute the VERL binding interface were also the most distorted regions of the crystal structure relative to what was observed in solution. While lysin has been the subject of extensive biochemical and evolutionary analyses for more than 30 years, this study highlights the enhanced insights gained from applying NMR approaches to rapidly evolving proteins.SignificanceThe fertilization of eggs by sperm is a critical biological process for nearly all sexually reproducing organisms to propagate their genetic information. Despite the importance of fertilization, the molecules that mediate egg-sperm interactions have been characterized for only a few species, and the biochemical mechanisms underlying these interactions are even less well understood. In the marine mollusk abalone, sperm lysin interacts with egg VERL in a species-specific manner to facilitate fertilization. In this report, we characterized the solution structure and molecular evolution of sperm lysin from red abalone (Haliotis rufescens), and identified the VERL binding interface as well as important lysin dimer interactions that have emerged as part of an incessant sexual arms race.

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SNP discovery and High Resolution Melting Analysis from massive transcriptome sequencing in the California red abalone Haliotis rufescens

Marine Genomics ◽

10.1016/j.margen.2012.12.003 ◽

2013 ◽

Vol 10 ◽

pp. 11-16 ◽

Cited By ~ 11

Author(s):

Valentina Valenzuela-Muñoz ◽

José Miguel Araya-Garay ◽

Cristian Gallardo-Escárate

Keyword(s):

High Resolution ◽

Transcriptome Sequencing ◽

High Resolution Melting ◽

High Resolution Melting Analysis ◽

Snp Discovery ◽

Haliotis Rufescens ◽

Red Abalone ◽

Melting Analysis

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Stereo Electron Microscopy Applied to High Resolution Freeze-Etch Replicas

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100068527 ◽

1970 ◽

Vol 28 ◽

pp. 306-307

Author(s):

L. Andrew Staehelin

Keyword(s):

Electron Microscopy ◽

Plastic Deformation ◽

High Resolution ◽

Smooth Surfaces ◽

Small Particles ◽

Membrane Surfaces ◽

Wide Acceptance ◽

New Information ◽

Number Of Particles ◽

Small Holes

Freeze-etched membranes usually appear as relatively smooth surfaces covered with numerous small particles and a few small holes (Fig. 1). In 1966 Branton (1“) suggested that these surfaces represent split inner mem¬brane faces and not true external membrane surfaces. His theory has now gained wide acceptance partly due to new information obtained from double replicas of freeze-cleaved specimens (2,3) and from freeze-etch experi¬ments with surface labeled membranes (4). While theses studies have fur¬ther substantiated the basic idea of membrane splitting and have shown clearly which membrane faces are complementary to each other, they have left the question open, why the replicated membrane faces usually exhibit con¬siderably fewer holes than particles. According to Branton's theory the number of holes should on the average equal the number of particles. The absence of these holes can be explained in either of two ways: a) it is possible that no holes are formed during the cleaving process e.g. due to plastic deformation (5); b) holes may arise during the cleaving process but remain undetected because of inadequate replication and microscope techniques.

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A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis

Genes ◽

10.3390/genes12020199 ◽

2021 ◽

Vol 12 (2) ◽

pp. 199

Author(s):

Xiaochun Zhang ◽

Huan Yu ◽

Qi Yang ◽

Ziwei Wang ◽

Ruocheng Xia ◽

...

Keyword(s):

High Resolution ◽

Dna Barcoding ◽

High Resolution Melting ◽

Its Region ◽

Its Sequencing ◽

Melting Temperatures ◽

Hrm Analysis ◽

National Drug ◽

Species Specific ◽

Its1 And Its2

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

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Bond-Breakage Mechanism in the Predissociation of theÃState ofN2O+: New Information from High Resolution Spectroscopy

Physical Review Letters ◽

10.1103/physrevlett.49.120 ◽

1982 ◽

Vol 49 (2) ◽

pp. 120-124 ◽

Cited By ~ 21

Author(s):

S. Abed ◽

M. Broyer ◽

M. Carré ◽

M. L. Gaillard ◽

M. Larzillière

Keyword(s):

High Resolution ◽

High Resolution Spectroscopy ◽

Bond Breakage ◽

New Information ◽

Breakage Mechanism

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High-resolution structures of inhibitor complexes of human indoleamine 2,3-dioxygenase 1 in a new crystal form

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18012955 ◽

2018 ◽

Vol 74 (11) ◽

pp. 717-724 ◽

Cited By ~ 10

Author(s):

Shukun Luo ◽

Ke Xu ◽

Shaoyun Xiang ◽

Jie Chen ◽

Chunyun Chen ◽

...

Keyword(s):

Drug Discovery ◽

High Resolution ◽

Crystal Structures ◽

Active Site ◽

Crystal Form ◽

Crystal Forms ◽

Cellular Processes ◽

Potential Target ◽

Indoleamine 2,3 Dioxygenase

Human indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme with important roles in many cellular processes and is a potential target for drug discovery against cancer and other diseases. Crystal structures of IDO1 in complex with various inhibitors have been reported. Many of these crystals belong to the same crystal form and most of the reported structures have resolutions in the range 3.2–2.3 Å. Here, three new crystal forms of human IDO1 obtained by introducing a surface mutation, K116A/K117A, distant from the active site are reported. One of these crystal forms diffracted to 1.5 Å resolution and can be readily used for soaking experiments to determine high-resolution structures of IDO1 in complex with the substrate tryptophan or inhibitors that coordinate the heme. In addition, this mutant was used to produce crystals of a complex with an inhibitor that targets the apo form of the enzyme under the same conditions; the structure of this complex was determined at 1.7 Å resolution. Overall, this mutant represents a robust platform for determining the structures of inhibitor and substrate complexes of IDO1 at high resolution.

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