Computational affinity maturation of camelid single-domain intrabodies against the nonamyloid component of alpha-synuclein

Sai Pooja Mahajan,Bunyarit Meksiriporn,Fernando A. Escobedo,David C. Butler,Matthew P. DeLisa,Ilkay Kocer,Dujduan Waraho-Zhmayev,Kevin B. Weyant,Anne Messer

doi:10.1038/s41598-018-35464-7

Abstract

Improving the affinity of protein-protein interactions is a challenging problem that is particularly important in the development of antibodies for diagnostic and clinical use. Here, we used structure-based computational methods to optimize the binding affinity of VHNAC1, a single-domain intracellular antibody (intrabody) from the camelid family that was selected for its specific binding to the nonamyloid component (NAC) of human α-synuclein (α-syn), a natively disordered protein, implicated in the pathogenesis of Parkinson’s disease (PD) and related neurological disorders. Specifically, we performed ab initio modeling that revealed several possible modes of VHNAC1 binding to the NAC region of α-syn as well as mutations that potentially enhance the affinity between these interacting proteins. While our initial design strategy did not lead to improved affinity, it ultimately guided us towards a model that aligned more closely with experimental observations, revealing a key residue on the paratope and the participation of H4 loop residues in binding, as well as confirming the importance of electrostatic interactions. The binding activity of the best intrabody mutant, which involved just a single amino acid mutation compared to parental VHNAC1, was significantly enhanced primarily through a large increase in association rate. Our results indicate that structure-based computational design can be used to successfully improve the affinity of antibodies against natively disordered and weakly immunogenic antigens such as α-syn, even in cases such as ours where crystal structures are unavailable.

Highlights

Α-Synuclein (α-syn) is a presynaptic neuronal protein that is pathologically linked to a number of neurodegenerative disorders collectively known as synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)[1,2,3,4]
The single-chain Fv intrabody NAC32 that is specific for amino acids 53–87 of α-syn comprising part of the nonamyloid component (NAC) region, was found to reduce the toxicity caused by the A53T mutant of α-syn in cultured cells[22]
The protein has an amphipathic N-terminus and acidic C-terminus that are separated by a central hydrophobic domain known as the NAC region

Summary

Introduction

Α-Synuclein (α-syn) is a presynaptic neuronal protein that is pathologically linked to a number of neurodegenerative disorders collectively known as synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)[1,2,3,4]. Because α-syn may play a central role in pathogenesis, reducing intracellular levels to prevent the abnormal misfolding, aggregation, and toxicity in vulnerable cells may serve as a potential therapeutic strategy for slowing the progression of PD and other synucleinopathies where misfolded proteins and subsequent protein aggregation appears to be an underlying factor[4] To achieve such an outcome, intracellular antibodies (intrabodies) have been proposed as a strategy for targeting and/or neutralizing different aberrant α-syn species[5,6,7,8]. The affinity of the hits (in the micromolar range) was deemed to be insufficient for biological activity In support of this notion, a tighter binding clone (Kd ≈ 30 nM) was isolated by affinity maturation using yeast display and found to efficiently block htt aggregation in cultured mammalian cells. Such approaches have been used by several groups to design mutations in antibodies that significantly improve their affinity towards antigen[35,36,37,38,39,40,41]

Methods

Results

Conclusion