Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein–cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin–γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens–epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone–client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone–client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin–client complexes could contribute to lens aging and presbyopia.

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30–40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell–engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Tau is a microtubule-associated protein that plays a major role in Alzheimer's disease (AD) and other tauopathies. Recent reports indicate that, in the presence of crowding agents, tau can undergo liquid–liquid phase separation (LLPS), forming highly dynamic liquid droplets. Here, using recombinantly expressed proteins, turbidimetry, fluorescence microscopy imaging, and fluorescence recovery after photobleaching (FRAP) assays, we show that the divalent transition metal zinc strongly promotes this process, shifting the equilibrium phase boundary to lower protein or crowding agent concentrations. We observed no tau LLPS-promoting effect for any other divalent transition metal ions tested, including Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. We also demonstrate that multiple zinc-binding sites on tau are involved in the LLPS-promoting effect and provide insights into the mechanism of this process. Zinc concentration is highly elevated in AD brains, and this metal ion is believed to be an important player in the pathogenesis of this disease. Thus, the present findings bring a new dimension to understanding the relationship between zinc homeostasis and the pathogenic process in AD and related neurodegenerative disorders.

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1–17, domains 1–17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all β-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1–17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed “templated folding,” whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Philip Newsholme
Dec 1, 2006; 55:S39-S47
Section II: The Muscle and Liver Connections

Ole Schmitz
Dec 1, 2004; 53:S233-S238
Section V: The Incretin Pathway

Barbara H. Braffett
May 1, 2020; 69:1000-1010
Complications

Sophie Rousset
Feb 1, 2004; 53:S130-S135
Section III: Mitochondria, Beta-Cell Function, and Type 2 Diabetes

Andrew S. Kimball
Sep 1, 2017; 66:2459-2471
Immunology and Transplantation

David J. Hodson
May 1, 2020; 69:864-866
Commentaries

Mandeep Bajaj
Nov 1, 2005; 54:3148-3153
Metabolism

D. Grahame Hardie
Jul 1, 2013; 62:2164-2172
Perspectives in Diabetes

Maria Sörhede Winzell
Dec 1, 2004; 53:S215-S219
Section V: The Incretin Pathway

Christer S. Andreassen
Mar 1, 2006; 55:806-812
Complications

Lena Eliasson
May 1, 2020; 69:804-812
Small Noncoding RNAs in Diabetes

Richard J. Johnson
Oct 1, 2013; 62:3307-3315
Perspectives in Diabetes

Mandeep Bajaj
Dec 1, 2012; 61:3078-3080
Commentary

Anne Jörns
Apr 1, 2020; 69:624-633
Islet Studies

Luigi Fontana
Apr 1, 2007; 56:1010-1013
Metabolism

Oct 1, 1992; 41:1368-1369
System[egrave] International (SI) Units Table

Jens Juul Holst
Dec 1, 2004; 53:S197-S204
Section V: The Incretin Pathway

Sarah M. Gray
Dec 1, 2014; 63:3992-3997
Perspectives in Diabetes

Mariam Alatrach
Apr 1, 2020; 69:681-688
Pathophysiology

Jennifer Vogel
May 1, 2020; 69:1032-1041
Pharmacology and Therapeutics

Marc Y. Donath
Dec 1, 2005; 54:S108-S113
Section III: Inflammation and beta-Cell Death

Ranjit Unnikrishnan
Jun 1, 2017; 66:1432-1442
Perspectives in Diabetes

Patrice D. Cani
Jun 1, 2008; 57:1470-1481
Metabolism

Mikael Knip
Dec 1, 2005; 54:S125-S136
Section IV: Polygenic Disease and Environment

Roy Taylor
Apr 1, 2012; 61:778-779
Commentary

Kristian Löbner
Mar 1, 2006; 55:792-797
Pathophysiology

Mario Luca Morieri
Apr 1, 2020; 69:771-783
Genetics/Genomes/Proteomics/Metabolomics

Juli Bai
Jun 1, 2019; 68:1099-1108
Perspectives in Diabetes

Errol B. Marliss
Feb 1, 2002; 51:S271-S283
Section 6: Pusatile and Phasic Insulin Release in Normal and Diabetic Men

Aldi T. Kraja
Apr 1, 2011; 60:1329-1339
Genetics

Meritxell Morró
May 1, 2020; 69:927-939
Islet Studies

Edwin A.M. Gale
Dec 1, 2002; 51:3353-3361
Perspectives in Diabetes

Daniel Batlle
Dec 1, 2010; 59:2994-2996
Commentaries

Mary C. Gannon
Sep 1, 2004; 53:2375-2382
Pathophysiology

Miriam Cnop
Dec 1, 2005; 54:S97-S107
Section III: Inflammation and beta-Cell Death

Patrice D. Cani
Jul 1, 2007; 56:1761-1772
Obesity Studies

Marlou L. Dirks
Oct 1, 2016; 65:2862-2875
Metabolism

Rhian M. Touyz
Jan 1, 2016; 65:19-21
Commentaries

Catherine Pihoker
Dec 1, 2005; 54:S52-S61
Section II: Type 1-Related Forms of Diabetes

Bruce B. Duncan
Jul 1, 2003; 52:1799-1805
Pathophysiology