Sterol A-ring plasticity in hedgehog protein cholesterolysis supports a primitive substrate selectivity mechanism

Ciulla, Daniel A.; Wagner, Andrew G.; Liu, Xinyue; Cooper, Courtney L.; Jorgensen, Michael T; Wang, Chunyu; Goyal, Puja; Banavali, Nilesh K.; Pezzullo, John L.; Giner, José‐Luis; Callahan, Brian P.

doi:10.1039/c8cc09729a

Cited by 9 publications

(12 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hh conjugates with cholesterol in vivo through this mechanism, however, other sterols can also be conjugated to Hh in vitro . Recent experimental data suggest different Hh autoprocessing activity for different sterol substitutions . RGATS simulations also offer an easy and inexpensive computational route to probe the differential binding and reactivity of these sterols.…”

Section: Discussionmentioning

confidence: 99%

The Mechanism of Cholesterol Modification of Hedgehog Ligand

Banavali

2019

J Comput Chem

Self Cite

View full text Add to dashboard Cite

Hedgehog (Hh) proteins are important components of signal transduction pathways involved in animal development, and their defects are implicated in carcinogenesis. Their N-terminal domain (HhN) acts as a signaling ligand, and their C-terminal domain (HhC) performs an autocatalytic function of cleaving itself away, while adding a cholesterol moiety to HhN. HhC has two sub-domains: a hedgehog/intein (hint) domain that primarily performs the autocatalytic activity, and a sterol-recognition region (SRR) that binds to cholesterol and properly positions it with respect to HhN. The three-dimensional details of this autocatalytic mechanism remain unknown, as does the structure of the precursor Hh protein. In this study, a complete cholesterol-bound precursor form of the drosophila Hh precursor is modeled using known crystal structures of HhN and the hint domain, and a hypothesized similarity of SRR to an unrelated but similar-sized cholesterol binding protein. The restrained geometries and topology switching (RGATS) strategy is then used to predict atomic-detail pathways for the full autocatalytic reaction starting from the precursor and ending in a cholesterol-linked HhN domain and a cleaved HhC domain. The RGATS explicit solvent simulations indicate the roles of individual HhC residues in facilitating the reaction, which can be confirmed through mutational experiments. These simulations also provide plausible structural models for the N/S acyl transfer intermediate and the product states of this reaction. This study thus provides a good framework for future computational and experimental studies to develop a full structural and dynamic understanding of Hh autoprocessing.

show abstract

Section: Discussionmentioning

confidence: 99%

The Mechanism of Cholesterol Modification of Hedgehog Ligand

Banavali

2019

J Comput Chem

Self Cite

View full text Add to dashboard Cite

show abstract

“…Hh autoprocessing occurs in following two steps ( Figure 1 ): 1) N-S Acyl Shift : C258 of HhC carries out a nucleophilic attack on the carbonyl of the last glycine (G-258) residue of HhN, resulting in a thioester intermediate. 2) Transesterification : The hydroxyl group of cholesterol non-covalently bound to the sterol recognition region (SRR) of HhC carries out a nucleophilic attack on the thioester, releasing HhN and linking it covalently to cholesterol ( Ciulla et al, 2019 ). Inteins, on the other hand, catalyze protein splicing processes in four steps ( Wall et al, 2021 ), with the first two steps very similar to Hh autoprocessing ( Koonin, 1995 ; Hall et al, 1997 ; Pietrokovski, 1998 ), except that the intramolecular transesterification in intein splicing is replaced by an intermolecular transesterification, namely cholesteroylation of HhN in Hh proteins ( Porter et al, 1996b ).…”

Section: Structural Mechanism Of Hedgehog Autoprocessingmentioning

confidence: 99%

“…Hh autoprocessing is important because it is at the origin of canonical Hh signaling, where it precedes all downstream signaling events ( Porter et al, 1996a ; Hall et al, 1997 ; Jiang and Paulus, 2010 ; Xie et al, 2014 ; Xie et al, 2015 ; Xie et al, 2016 ; Zhang et al, 2019 ; Zhao et al, 2019 ; Smith et al, 2020 ), and is unique to Hh proteins. Although it lies at the very origin of Hh signaling, there are only a few structural/mechanistic studies ( Owen et al, 2015a ; Owen et al, 2015b ; Callahan and Wang, 2015 ; Bordeau et al, 2016 ; Ciulla et al, 2018 ; Ciulla et al, 2019 ; Zhang et al, 2019 ; Zhao et al, 2019 ; Smith et al, 2020 ), compared to the great number of studies of downstream components, such as PTCH ( Ingham et al, 1991 ; Chen and Struhl, 1996 ; Sidransky, 1996 ; Kallassy et al, 1997 ; Xie et al, 1997 ; Zedan et al, 2001 ; Shao et al, 2006 ; Lorberbaum et al, 2016 ; Tukachinsky et al, 2016 ; Zhang et al, 2018 ; Abd Elrhman and Ebian, 2019 ; Kinnebrew et al, 2021 ), SMO ( Xie et al, 2014 ; Owen et al, 2015a ; Owen et al, 2015b ; Callahan and Wang, 2015 ; Xie et al, 2015 ; Xie et al, 2016 ; Zhang et al, 2019 ; Zhao et al, 2019 ; Smith et al, 2020 ), and GLI ( Lauth et al, 2007 ; Kim et al, 2010 ; Maun et al, 2010 ; Beauchamp et al, 2011 ; Pan et al, 2012 ; Long et al, 2016 ; Xiao et al, 2017 ; Kowatsch et al, 2019 ; Liu et al, 2019 ; Quaglio et al, 2020 ; Tran et al, 2020 ). In some context with direct cell-to-cell contact, the unprocessed full-length Hh protein is reported to have signalin...…”

Section: Introductionmentioning

confidence: 99%

Hedgehog Autoprocessing: From Structural Mechanisms to Drug Discovery

Kandel

Wang

2022

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

Hedgehog (Hh) signaling plays pivotal roles in embryonic development. In adults, Hh signaling is mostly turned off but its abnormal activation is involved in many types of cancer. Hh signaling is initiated by the Hh ligand, generated from the Hh precursor by a specialized autocatalytic process called Hh autoprocessing. The Hh precursor consists of an N-terminal signaling domain (HhN) and a C-terminal autoprocessing domain (HhC). During Hh autoprocessing, the precursor is cleaved between N- and C-terminal domain followed by the covalent ligation of cholesterol to the last residue of HhN, which subsequently leads to the generation of Hh ligand for Hh signaling. Hh autoprocessing is at the origin of canonical Hh signaling and precedes all downstream signaling events. Mutations in the catalytic residues in HhC can lead to congenital defects such as holoprosencephaly (HPE). The aim of this review is to provide an in-depth summary of the progresses and challenges towards an atomic level understanding of the structural mechanisms of Hh autoprocessing. We also discuss drug discovery efforts to inhibit Hh autoprocessing as a new direction in cancer therapy.

show abstract

“…We have found that the Drosophila melanogaster HhC has broad substrate tolerance, maintaining robust bioconjugation activity toward heterologous N-terminal substrate proteins and catalyzing ligation with sterols of varying structure and chemical appendages, including oligonucleotides. [15,16] For E-beacons, we fused this promiscuous HhC to the C-terminus of the Nluc enzyme (Supporting Information).…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid**

et al. 2021

Self Cite

View full text Add to dashboard Cite

Enzymatic beacons, or E-beacons, are 1 : 1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming ssDNA equipped with a dark quencher. We prepared E-beacons biocatalytically using HhC, the promiscuous Hedgehog C-terminal protein-cholesterol ligase. HhC attached nanoluciferase site-specifically to mono-sterylated hairpin oligonucleotides, called steramers. Three E-beacon dark quenchers were evaluated: Iowa Black, Onyx-A, and dabcyl. Each quencher enabled sensitive, sequence-specific nucleic acid detection through enhanced E-beacon bioluminescence upon target hybridization. We assembled prototype dabcyl-quenched Ebeacons specific for SARS-CoV-2. Targeting the E484 codon of the virus Spike protein, E-beacons (80 × 10 À 12 M) reported wildtype SARS-CoV-2 nucleic acid at � 1 × 10 À 9 M by increased bioluminescence of 8-fold. E-beacon prepared for the SARS-CoV-2 E484K variant functioned with similar sensitivity. Both Ebeacons could discriminate their target from the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with mismatch specificity, E-beacons are two to three orders of magnitude more sensitive than synthetic molecular beacons.

show abstract

Sterol A-ring plasticity in hedgehog protein cholesterolysis supports a primitive substrate selectivity mechanism

Abstract: The enzymatic agent of hedgehog protein cholesterolysis accommodates substrate sterols with undersized, oversized and distorted ring systems relative to cholesterol.

Cited by 9 publications

References 31 publications

The Mechanism of Cholesterol Modification of Hedgehog Ligand

The Mechanism of Cholesterol Modification of Hedgehog Ligand

Hedgehog Autoprocessing: From Structural Mechanisms to Drug Discovery

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid**

Contact Info

Product

Resources

About