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A pivotal role for a conserved bulky residue at the ?1-helix of the ?I integrin domain in ligand binding.

ABSTRACT: The ligand-binding ?I and ?I domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both ?I and ?I domains, the ?1- and ?7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the ?1-helix of ?I helps maintain the resting ?I conformation, whereas the homologous position in the ?I ?1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using ?L?2 integrin as a model, we found that the residue volume at the Phe position in the ?1-helix is critical for ?L?2 activation because trimming the Phe by small amino acid substitutions abolished ?L?2 binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for ?M?2 integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of ?6-?7 loop and ?7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between ?L ?I and ?2 ?I could be uncoupled because the ?2 extension and headpiece opening could occur independently of the ?I activation. Reciprocally, the ?I activation does not inevitably lead to the conformational changes of the ?2 subunit. Such loose linkage between the ?I and ?I is attributed to the ?I flexibility and could accommodate the ?L?2-mediated rolling adhesion of leukocytes.

SUBMITTER: Wang Z

PROVIDER: S-EPMC5733610 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding.

Wang Zhengli Z Thinn Aye Myat Myat AMM Zhu Jieqing J

The Journal of biological chemistry 20171027 50

The ligand-binding βI and αI domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both βI and αI domains, the α1- and α7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the α1-helix of βI helps maintain the resting βI conformation, whereas the homologous position in the αI α1-helix contains a conse ...[more]

PMID: 29079572

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Project description:BACKGROUND: The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded ? sheet and three long ? helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?" METHODS: We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded ? sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3 day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. RESULTS: This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional "domain" found to date for the matrixin family. CONCLUSIONS: The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.

Dataset Information

A pivotal role for a conserved bulky residue at the ?1-helix of the ?I integrin domain in ligand binding.

Publications

A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding.

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