A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

De-la-Torre, Pedro; Choudhary, Deepanshu; Araya-Secchi, Raúl; Narui, Yoshie; Sotomayor, Marcos

doi:10.1016/j.bpj.2018.11.010

Cited by 29 publications

(69 citation statements)

References 147 publications

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“…The elastic response of MAD12 in the context of stretching simulations of the monomeric hs and mm PCDH15 EC1-MAD12 + CDH23 EC1-2/3 complexes is consistent with simulations stretching the dimeric mm PCDH15 EC9-MAD12 (SI Appendix, Fig. S12 D and E and Movie S3) and our previous simulations of ss PCDH15 EC10-MAD12 (28). Our new simulations including the entire monomeric PCDH15 ectodomain predict soft elasticity and unfolding of MAD12 before unbinding of the cadherin handshake at fast stretching speeds.…”

Section: Pcdh15 Ectodomain C-terminal Structures Reveal Kinks and Cissupporting

confidence: 88%

“…To gain further insights into the function of specific repeats and to build a complete model of PCDH15, we worked with multiple WT (hs NP_001136235.1; mm NP_075604.2) and mutated protein fragments (rationale for mutations are explained throughout the text). Successful expression, purification, crystallization, and (28,31,32). All structures are in surface representation, with PCDH15 fragments in purple, CDH23 EC1-2 in blue, Ca 2+ ions in green, and glycosylation sugars in red and yellow.…”

Section: Resultsmentioning

confidence: 99%

“…( C ) Schematics of PCDH15 ( Left ) and structures of PCDH15 fragments ( Right ) used to build models of the entire PCDH15 ectodomain. PDB codes are indicated for all 11 structures presented here, with codes in parentheses for three additional structures presented elsewhere ( 28 , 31 , 32 ). All structures are in surface representation, with PCDH15 fragments in purple, CDH23 EC1-2 in blue, Ca 2+ ions in green, and glycosylation sugars in red and yellow.…”

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confidence: 99%

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Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception

Choudhary

Narui

Neel

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical stimuli originating from an extraordinarily broad range of sound frequencies and intensities or from head movements. Integral to these processes is the tip-link protein complex, which conveys force to open the inner-ear transduction channels that mediate sensory perception. Protocadherin-15 and cadherin-23, two atypically large cadherins with 11 and 27 extracellular cadherin (EC) repeats, are involved in deafness and balance disorders and assemble as parallel homodimers that interact to form the tip link. Here we report the X-ray crystal structure of a protocadherin-15 + cadherin-23 heterotetrameric complex at 2.9-Å resolution, depicting a parallel homodimer of protocadherin-15 EC1-3 molecules forming an antiparallel complex with two cadherin-23 EC1-2 molecules. In addition, we report structures for 10 protocadherin-15 fragments used to build complete high-resolution models of the monomeric protocadherin-15 ectodomain. Molecular dynamics simulations and validated crystal contacts are used to propose models for the complete extracellular protocadherin-15 parallel homodimer and the tip-link bond. Steered molecular dynamics simulations of these models suggest conditions in which a structurally diverse and multimodal protocadherin-15 ectodomain can act as a stiff or soft gating spring. These results reveal the structural determinants of tip-link–mediated inner-ear sensory perception and elucidate protocadherin-15’s structural and adhesive properties relevant in disease.

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Section: Pcdh15 Ectodomain C-terminal Structures Reveal Kinks and Cissupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception

Choudhary

Narui

Neel

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Another contribution comes from the ability of individual components to rebind after they rupture, dynamically maintaining the overall connection. Since CDH23 and PCDH15 both form strong cis-dimers in-vitro and in-vivo, facilitated by bonds along their ectodomains 6,7,[28][29][30][31] , the tip link may increase its lifetime through rebinding if opposing PCDH15 and CDH23 EC1-2 domains are kept in sufficiently close spatial proximity, creating a high effective local concentration and a fast on-rate.…”

Section: Main Textmentioning

confidence: 99%

“…Since our model predicts that the mechanical properties of tip-link cadherins significantly affect avidity, we made structural modifications to the tip link proteins to test this effect. Cis-dimerization links PCDH15 and CDH23 laterally at several points along their length 6,7,[28][29][30][31] , and may act to stabilize Ceff under force. We truncated PCDH15 and CDH23 to their first five EC domains in an effort to disrupt full cis-dimerization, and used single-molecule force spectroscopy to determine the effects on bond strength ( Fig.…”

Section: Main Textmentioning

confidence: 99%

The Dynamic Strength of the Hair-Cell Tip Link Reveals Mechanisms of Hearing and Deafness

Mulhall

Ward

Yang

et al. 2019

Preprint

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Our senses of hearing and balance rely on the extraordinarily sensitive molecular machinery of the inner ear to convert deflections as small as the width of a single carbon atom 1,2 into electrical signals that the brain can process 3 . In humans and other vertebrates, transduction is mediated by hair cells 4 , where tension on tip links conveys force to mechanosensitive ion channels 5 . Each tip link comprises two helical filaments of atypical cadherins bound at their N-termini through two unique adhesion bonds 6-8 . Tip links must be strong enough to maintain a connection to the mechanotransduction channel under the dynamic forces exerted by sound or head movement-yet might also act as mechanical circuit breakers, releasing under extreme conditions to preserve the delicate structures within the hair cell. Previous studies have argued that this connection is exceptionally static, disrupted only by harsh chemical conditions or loud sound 9-12 . However, no direct mechanical measurements of the full tip-link connection have been performed. Here we describe the dynamics of the tiplink connection at single-molecule resolution and show how avidity conferred by its double stranded architecture enhances mechanical strength and lifetime, yet still enables it to act as a dynamic mechanical circuit breaker. We also show how the dynamic strength of the connection is facilitated by strong cisdimerization and tuned by extracellular Ca 2+ , and we describe the unexpected etiology of a hereditary human deafness mutation. Remarkably, the connection is several thousand times more dynamic than previously thought, challenging current assumptions about tip-link stability and turnover rate, and providing insight into how the mechanotransduction apparatus conveys mechanical information. Our results reveal fundamental mechanisms that underlie mechanoelectric transduction in the inner ear, and provide a foundation for studying multi-component linkages in other biological systems.

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PKHD1L1, a gene involved in the stereocilia coat, causes autosomal recessive nonsyndromic hearing loss

Redfield,

De-la-Torre,

Zamani

et al. 2024

Hum. Genet.

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Identification of genes associated with nonsyndromic hearing loss is a crucial endeavor given the substantial number of individuals who remain without a diagnosis after even the most advanced genetic testing. PKHD1L1 was established as necessary for the formation of the cochlear hair-cell stereociliary coat and causes hearing loss in mice and zebrafish when mutated. We sought to determine if biallelic variants in PKHD1L1 also cause hearing loss in humans. Exome sequencing was performed on DNA of four families segregating autosomal recessive nonsyndromic sensorineural hearing loss. Compound heterozygous p.[(Gly129Ser)];p.[(Gly1314Val)] and p.[(Gly605Arg)];p[(Leu2818TyrfsTer5)], homozygous missense p.(His2479Gln) and nonsense p.(Arg3381Ter) variants were identified in PKHD1L1 that were predicted to be damaging using in silico pathogenicity prediction methods. In vitro functional analysis of two missense variants was performed using purified recombinant PKHD1L1 protein fragments. We then evaluated protein thermodynamic stability with and without the missense variants found in one of the families and performed a minigene splicing assay for another variant. In silico molecular modeling using AlphaFold2 and protein sequence alignment analysis were carried out to further explore potential variant effects on structure. In vitro functional assessment indicated that both engineered PKHD1L1 p.(Gly129Ser) and p.(Gly1314Val) mutant constructs significantly reduced the folding and structural stabilities of the expressed protein fragments, providing further evidence to support pathogenicity of these variants. Minigene assay of the c.1813G>A p.(Gly605Arg) variant, located at the boundary of exon 17, revealed exon skipping leading to an in-frame deletion of 48 amino acids. In silico molecular modeling exposed key structural features that might suggest PKHD1L1 protein destabilization. Multiple lines of evidence collectively associate PKHD1L1 with nonsyndromic mild–moderate to severe sensorineural hearing loss. PKHD1L1 testing in individuals with mild–moderate hearing loss may identify further affected families.

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A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

Cited by 29 publications

References 147 publications

Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception

Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception

The Dynamic Strength of the Hair-Cell Tip Link Reveals Mechanisms of Hearing and Deafness

PKHD1L1, a gene involved in the stereocilia coat, causes autosomal recessive nonsyndromic hearing loss

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