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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Chemical Biology".

Deadline for manuscript submissions: closed (15 July 2020) | Viewed by 102040

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Prof. Dr. Silvano Geremia

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CEB Centre of Excellence in Biocrystallography, Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy
Interests: crystallography; structural biology; Vitamin B12 proteins; redox-proteins; drug delivery; diagnostics; metals in medicine; bioinorganic chemistry; supramolecular chemistry; nanostructures
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Dear Colleagues,

Structural biology is a relatively young science, initiated about 60 years ago with the elucidation of the first three-dimensional crystal structure of myoglobin. Protein crystallography continues to develop vigorously and today there are over 120,000 structures deposited at the Protein Data Bank (PDB), 90% of which are from X-ray data. The technological developments behind this rapid growth involves all crucial steps of the pipeline “from gene to structure”: from the developments of wet lab technologies, including recombinant DNA techniques, protein purification and crystallization; to innovative hardware technology, for example brilliant light sources such as synchrotrons and X-ray free electron lasers (XFEL) together with high-frame-rate and ultra-sensitive detectors. An important impetus to protein crystallography has also been provided by soft technology: theoretical foundations, computational algorithm, and software development. The bio-crystallography integrated with cryo-electron microscopy is a major research trend in structural biology and the present Special Issue is aimed at covering frontier technologies and methodologies in protein crystallography, as well as their novel applications.

Prof. Dr. Silvano Geremia
Guest Editor

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Keywords

X-ray crystallography
recombinant protein overexpression
protein purification
protein crystallization
cryo-crystallography
radiation sources
X-ray detectors
structural biology software
high-throughput crystallography
structural biology

Published Papers (14 papers)

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Research

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10 pages, 1285 KiB

Open AccessArticle

High-Throughput Crystallization Pipeline at the Crystallography Core Facility of the Institut Pasteur

by Patrick Weber, Cédric Pissis, Rafael Navaza, Ariel E. Mechaly, Frederick Saul, Pedro M. Alzari and Ahmed Haouz

Molecules 2019, 24(24), 4451; https://doi.org/10.3390/molecules24244451 - 05 Dec 2019

Cited by 37 | Viewed by 4189

Abstract

The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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14 pages, 2955 KiB

Open AccessArticle

Structural Basis for the Regulation of PPARγ Activity by Imatinib

by Jun Young Jang, Hyun-Jung Kim and Byung Woo Han

Molecules 2019, 24(19), 3562; https://doi.org/10.3390/molecules24193562 - 01 Oct 2019

Cited by 9 | Viewed by 3235

Abstract

Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2′ and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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13 pages, 3656 KiB

Open AccessArticle

Sub-Atomic Resolution Crystal Structures Reveal Conserved Geometric Outliers at Functional Sites

by Saara Laulumaa and Petri Kursula

Molecules 2019, 24(17), 3044; https://doi.org/10.3390/molecules24173044 - 22 Aug 2019

Cited by 4 | Viewed by 3343

Abstract

Myelin protein 2 (P2) is a peripheral membrane protein of the vertebrate nervous system myelin sheath, having possible roles in both lipid transport and 3D molecular organization of the multilayered myelin membrane. We extended our earlier crystallographic studies on human P2 and refined its crystal structure at an ultrahigh resolution of 0.72 Å in perdeuterated form and 0.86 Å in hydrogenated form. Characteristic differences in C–H…O hydrogen bond patterns were observed between extended β strands, kinked or ending strands, and helices. Often, side-chain C–H groups engage in hydrogen bonding with backbone carbonyl moieties. The data highlight several amino acid residues with unconventional conformations, including both bent aromatic rings and twisted guanidinium groups on arginine side chains, as well as non-planar peptide bonds. In two locations, such non-ideal conformations cluster, providing proof of local functional strain. Other ultrahigh-resolution protein structures similarly contain chemical groups, which break planarity rules. For example, in Src homology 3 (SH3) domains, a conserved bent aromatic residue is observed near the ligand binding site. Fatty acid binding protein (FABP) 3, belonging to the same family as P2, has several side chains and peptide bonds bent exactly as those in P2. We provide a high-resolution snapshot on non-ideal conformations of amino acid residues under local strain, possibly relevant to biological function. Geometric outliers observed in ultrahigh-resolution protein structures are real and likely relevant for ligand binding and conformational changes. Furthermore, the deuteration of protein and/or solvent are promising variables in protein crystal optimization. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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28 pages, 5966 KiB

Open AccessArticle

Structural Characterization of Arabidopsis thaliana NAP1-Related Protein 2 (AtNRP2) and Comparison with Its Homolog AtNRP1

by Ashish Kumar, Ajit Kumar Singh, Ruchir Chandrakant Bobde and Dileep Vasudevan

Molecules 2019, 24(12), 2258; https://doi.org/10.3390/molecules24122258 - 17 Jun 2019

Cited by 5 | Viewed by 3931

Abstract

Nucleosome Assembly Protein (NAP) is a highly conserved family of histone chaperones present in yeast, animals, and plants. Unlike other organisms, plants possess an additional class of proteins in its NAP family, known as the NAP1-related proteins or NRP. Arabidopsis thaliana possesses two NRP isoforms, namely AtNRP1 and AtNRP2, that share 87% sequence identity. Both AtNRP1 and AtNRP2 get expressed in all the plant tissues. Most works in the past, including structural studies, have focused on AtNRP1. We wanted to do a comparative study of the two proteins to find why the plant would have two very similar proteins and whether there is any difference between the two for their structure and function as histone chaperones. Here we report the crystal structure of AtNRP2 and a comparative analysis of its structural architecture with other NAP family proteins. The crystal structure of AtNRP2 shows it to be a homodimer, with its fold similar to that of other structurally characterized NAP family proteins. Although AtNRP1 and AtNRP2 have a similar fold, upon structural superposition, we find an offset in the dimerization helix of the two proteins. We evaluated the stability, oligomerization status, and histone chaperoning properties of the two proteins, for a comparison. The thermal melting experiments suggest that AtNRP2 is more stable than AtNRP1 at higher temperatures. In addition, electrophoretic mobility shift assay and isothermal titration calorimetry experiments suggest histone binding ability of AtNRP2 is higher than that of AtNRP1. Overall, these results provide insights about the specific function and relevance of AtNRP2 in plants through structural and biophysical studies. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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16 pages, 2720 KiB

Open AccessArticle

Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II

by Michelle S. Miller, Sweta Maheshwari, Wuxian Shi, Yuan Gao, Nam Chu, Alexei S. Soares, Philip A. Cole, L. Mario Amzel, Martin R. Fuchs, Jean Jakoncic and Sandra B. Gabelli

Molecules 2019, 24(3), 496; https://doi.org/10.3390/molecules24030496 - 30 Jan 2019

Cited by 11 | Viewed by 3897

Abstract

Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II—AMX and FMX—deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins—Akt1, PI3Kα, and CDP-Chase—to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or “raster-collect”, a more automated approach for a larger number of crystals (using CDP-Chase as an example). Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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Review

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24 pages, 3654 KiB

Open AccessReview

Atomic Details of Carbon-Based Nanomolecules Interacting with Proteins

by Luigi Di Costanzo and Silvano Geremia

Molecules 2020, 25(15), 3555; https://doi.org/10.3390/molecules25153555 - 04 Aug 2020

Cited by 15 | Viewed by 4506

Abstract

Since the discovery of fullerene, carbon-based nanomolecules sparked a wealth of research across biological, medical and material sciences. Understanding the interactions of these materials with biological samples at the atomic level is crucial for improving the applications of nanomolecules and address safety aspects concerning their use in medicine. Protein crystallography provides the interface view between proteins and carbon-based nanomolecules. We review forefront structural studies of nanomolecules interacting with proteins and the mechanism underlying these interactions. We provide a systematic analysis of approaches used to select proteins interacting with carbon-based nanomolecules explored from the worldwide Protein Data Bank (wwPDB) and scientific literature. The analysis of van der Waals interactions from available data provides important aspects of interactions between proteins and nanomolecules with implications on functional consequences. Carbon-based nanomolecules modulate protein surface electrostatic and, by forming ordered clusters, could modify protein quaternary structures. Lessons learned from structural studies are exemplary and will guide new projects for bioimaging tools, tuning of intrinsically disordered proteins, and design assembly of precise hybrid materials. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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18 pages, 1041 KiB

Open AccessFeature PaperReview

Protein X-ray Crystallography and Drug Discovery

by Laurent Maveyraud and Lionel Mourey

Molecules 2020, 25(5), 1030; https://doi.org/10.3390/molecules25051030 - 25 Feb 2020

Cited by 108 | Viewed by 16489

Abstract

With the advent of structural biology in the drug discovery process, medicinal chemists gained the opportunity to use detailed structural information in order to progress screening hits into leads or drug candidates. X-ray crystallography has proven to be an invaluable tool in this respect, as it is able to provide exquisitely comprehensive structural information about the interaction of a ligand with a pharmacological target. As fragment-based drug discovery emerged in the recent years, X-ray crystallography has also become a powerful screening technology, able to provide structural information on complexes involving low-molecular weight compounds, despite weak binding affinities. Given the low numbers of compounds needed in a fragment library, compared to the hundreds of thousand usually present in drug-like compound libraries, it now becomes feasible to screen a whole fragment library using X-ray crystallography, providing a wealth of structural details that will fuel the fragment to drug process. Here, we review theoretical and practical aspects as well as the pros and cons of using X-ray crystallography in the drug discovery process. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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13 pages, 910 KiB

Open AccessReview

Macromolecular Nanocrystal Structural Analysis with Electron and X-Rays: A Comparative Review

by Krishna P. Khakurel, Borislav Angelov and Jakob Andreasson

Molecules 2019, 24(19), 3490; https://doi.org/10.3390/molecules24193490 - 26 Sep 2019

Cited by 5 | Viewed by 4537

Abstract

Crystallography has long been the unrivaled method that can provide the atomistic structural models of macromolecules, using either X-rays or electrons as probes. The methodology has gone through several revolutionary periods, driven by the development of new sources, detectors, and other instrumentation. Novel sources of both X-ray and electrons are constantly emerging. The increase in brightness of these sources, complemented by the advanced detection techniques, has relaxed the traditionally strict need for large, high quality, crystals. Recent reports suggest high-quality diffraction datasets from crystals as small as a few hundreds of nanometers can be routinely obtained. This has resulted in the genesis of a new field of macromolecular nanocrystal crystallography. Here we will make a brief comparative review of this growing field focusing on the use of X-rays and electrons sources. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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30 pages, 4988 KiB

Open AccessReview

Structural Insights into the Regulation Mechanism of Small GTPases by GEFs

by Sachiko Toma-Fukai and Toshiyuki Shimizu

Molecules 2019, 24(18), 3308; https://doi.org/10.3390/molecules24183308 - 11 Sep 2019

Cited by 31 | Viewed by 9778

Abstract

Small GTPases are key regulators of cellular events, and their dysfunction causes many types of cancer. They serve as molecular switches by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. GTPases are deactivated by GTPase-activating proteins (GAPs) and are activated by guanine-nucleotide exchange factors (GEFs). The intrinsic GTP hydrolysis activity of small GTPases is generally low and is accelerated by GAPs. GEFs promote GDP dissociation from small GTPases to allow for GTP binding, which results in a conformational change of two highly flexible segments, called switch I and switch II, that enables binding of the gamma phosphate and allows small GTPases to interact with downstream effectors. For several decades, crystal structures of many GEFs and GAPs have been reported and have shown tremendous structural diversity. In this review, we focus on the latest structural studies of GEFs. Detailed pictures of the variety of GEF mechanisms at atomic resolution can provide insights into new approaches for drug discovery. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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20 pages, 2036 KiB

Open AccessReview

Normal Mode Analysis as a Routine Part of a Structural Investigation

by Jacob A. Bauer, Jelena Pavlović and Vladena Bauerová-Hlinková

Molecules 2019, 24(18), 3293; https://doi.org/10.3390/molecules24183293 - 10 Sep 2019

Cited by 43 | Viewed by 8374

Abstract

Normal mode analysis (NMA) is a technique that can be used to describe the flexible states accessible to a protein about an equilibrium position. These states have been shown repeatedly to have functional significance. NMA is probably the least computationally expensive method for studying the dynamics of macromolecules, and advances in computer technology and algorithms for calculating normal modes over the last 20 years have made it nearly trivial for all but the largest systems. Despite this, it is still uncommon for NMA to be used as a component of the analysis of a structural study. In this review, we will describe NMA, outline its advantages and limitations, explain what can and cannot be learned from it, and address some criticisms and concerns that have been voiced about it. We will then review the most commonly used techniques for reducing the computational cost of this method and identify the web services making use of these methods. We will illustrate several of their possible uses with recent examples from the literature. We conclude by recommending that NMA become one of the standard tools employed in any structural study. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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37 pages, 4689 KiB

Open AccessReview

Plant Leucine-Rich Repeat Receptor Kinase (LRR-RK): Structure, Ligand Perception, and Activation Mechanism

by Sayan Chakraborty, Brian Nguyen, Syed Danyal Wasti and Guozhou Xu

Molecules 2019, 24(17), 3081; https://doi.org/10.3390/molecules24173081 - 25 Aug 2019

Cited by 47 | Viewed by 10386

Abstract

In recent years, secreted peptides have been recognized as essential mediators of intercellular communication which governs plant growth, development, environmental interactions, and other mediated biological responses, such as stem cell homeostasis, cell proliferation, wound healing, hormone sensation, immune defense, and symbiosis, among others. Many of the known secreted peptide ligand receptors belong to the leucine-rich repeat receptor kinase (LRR-RK) family of membrane integral receptors, which contain more than 200 members within Arabidopsis making it the largest family of plant receptor kinases (RKs). Genetic and biochemical studies have provided valuable data regarding peptide ligands and LRR-RKs, however, visualization of ligand/LRR-RK complex structures at the atomic level is vital to understand the functions of LRR-RKs and their mediated biological processes. The structures of many plant LRR-RK receptors in complex with corresponding ligands have been solved by X-ray crystallography, revealing new mechanisms of ligand-induced receptor kinase activation. In this review, we briefly elaborate the peptide ligands, and aim to detail the structures and mechanisms of LRR-RK activation as induced by secreted peptide ligands within plants. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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16 pages, 4336 KiB

Open AccessReview

Molecular Interactions of Antibody Drugs Targeting PD-1, PD-L1, and CTLA-4 in Immuno-Oncology

by Hyun Tae Lee, Sang Hyung Lee and Yong-Seok Heo

Molecules 2019, 24(6), 1190; https://doi.org/10.3390/molecules24061190 - 26 Mar 2019

Cited by 169 | Viewed by 15793

Abstract

Cancer cells can evade immune surveillance through the molecular interactions of immune checkpoint proteins, including programmed death 1 (PD-1), PD-L1, and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Since 2011, the FDA-approved antibody drugs ipilimumab (Yervoy^®), nivolumab (Opdivo^®), pembrolizumab (Keytruda^®), cemiplimab (Libtayo^®), atezolizumab (Tecentriq^®), durvalumab (Imfinzi^®), and avelumab (Bavencio^®), which block the immune checkpoint proteins, have brought about a significant breakthrough in the treatment of a wide range of cancers, as they can induce durable therapeutic responses. In recent years, crystal structures of the antibodies against PD-1, PD-L1, and CTLA-4 have been reported. In this review, we describe the latest structural studies of these monoclonal antibodies and their interactions with the immune checkpoint proteins. A comprehensive analysis of the interactions of these immune checkpoint blockers can provide a better understanding of their therapeutic mechanisms of action. The accumulation of these structural studies would provide a basis that is essential for the rational design of next-generation therapies in immuno-oncology. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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16 pages, 6566 KiB

Open AccessReview

Approaches to the Structure-Based Design of Antivirulence Drugs: Therapeutics for the Post-Antibiotic Era

by Nolan Neville and Zongchao Jia

Molecules 2019, 24(3), 378; https://doi.org/10.3390/molecules24030378 - 22 Jan 2019

Cited by 19 | Viewed by 5161

Abstract

The alarming rise of multidrug-resistant bacterial strains, coupled with decades of stagnation in the field of antibiotic development, necessitates exploration of new therapeutic approaches to treat bacterial infections. Targeting bacterial virulence is an attractive alternative to traditional antibiotics in that this approach disarms pathogens that cause human diseases, without placing immediate selective pressure on the target bacterium or harming commensal species. The growing number of validated virulence protein targets for which structural information has been obtained, along with advances in computational power and screening algorithms, make the rational design of antivirulence drugs a promising avenue to explore. Here, we review the principles of structure-based drug design and the exciting opportunities this technique presents for antivirulence drug discovery. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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16 pages, 2536 KiB

Open AccessReview

A Brief History of Charcot-Leyden Crystal Protein/Galectin-10 Research

by Jiyong Su

Molecules 2018, 23(11), 2931; https://doi.org/10.3390/molecules23112931 - 09 Nov 2018

Cited by 41 | Viewed by 6970

Abstract

Eosinophils are present in tissues, such as the respiratory tract, spleen, lymph nodes and blood vessels. The significant presence of eosinophils in these tissues are associated with various diseases, including asthma, allergies, acute myeloid leukemia, etc. Charcot-Leyden crystal protein/galectin-10 is overexpressed in eosinophils and has also been identified in basophils and macrophages. In human body, this protein could spontaneously form Charcot-Leyden crystal in lymphocytes or in the lysates of lymphocytes. At present, the role of Charcot-Leyden crystal protein/galectin-10 in lymphocytes is not fully understood. This review summarizes research progress on Charcot-Leyden crystal protein/galectin-10, with emphasis on its history, cellular distributions, relations to diseases, structures and ligand binding specificity. Full article

(This article belongs to the Special Issue Frontier of Protein Crystallography)

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