1. |
Su SC,
Lin CC,
Tai HC,
Chang MY,
Ho MR,
Babu CS,
Liao JH,
Wu SH,
Chang YC,
Lim C,
Chang CI,
( 2016 ) Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease. PMID : 27041593 : DOI : 10.1016/j.str.2016.03.003 Abstract >>
The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and performs ATP-dependent proteolysis. However, it is also found that LonA can carry out Mg(2+)-dependent degradation of unfolded protein substrate in an ATP-independent manner. Here we show that in the presence of Mg(2+) LonA forms a non-secluded hexameric barrel with prominent openings, which explains why Mg(2+)-activated LonA can operate as a diffusion-based chambered protease to degrade unstructured protein and peptide substrates efficiently in the absence of ATP. A 1.85 ? crystal structure of Mg(2+)-activated protease domain reveals Mg(2+)-dependent remodeling of a substrate-binding loop and a potential metal-binding site near the Ser-Lys catalytic dyad, supported by biophysical binding assays and molecular dynamics simulations. Together, these findings reveal the specific roles of Mg(2+) in the molecular assembly and activation of LonA.
|
2. |
Lin CC,
Su SC,
Su MY,
Liang PH,
Feng CC,
Wu SH,
Chang CI,
( 2016 ) Structural Insights into the Allosteric Operation of the Lon AAA+ Protease. PMID : 27041592 : DOI : 10.1016/j.str.2016.03.001 Abstract >>
The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine.
|
3. |
Liao JH,
Ihara K,
Kuo CI,
Huang KF,
Wakatsuki S,
Wu SH,
Chang CI,
( 2013 ) Structures of an ATP-independent Lon-like protease and its complexes with covalent inhibitors. PMID : 23897463 : DOI : 10.1107/S0907444913008214 Abstract >>
The Lon proteases are a unique family of chambered proteases with a built-in AAA+ (ATPases associated with diverse cellular activities) module. Here, crystal structures of a unique member of the Lon family with no intrinsic ATPase activity in the proteolytically active form are reported both alone and in complexes with three covalent inhibitors: two peptidomimetics and one derived from a natural product. This work reveals the unique architectural features of an ATP-independent Lon that selectively degrades unfolded protein substrates. Importantly, these results provide mechanistic insights into the recognition of inhibitors and polypeptide substrates within the conserved proteolytic chamber, which may aid the development of specific Lon-protease inhibitors.
|
4. |
Li JK,
Liao JH,
Li H,
Kuo CI,
Huang KF,
Yang LW,
Wu SH,
Chang CI,
( 2013 ) The N-terminal substrate-recognition domain of a LonC protease exhibits structural and functional similarity to cytosolic chaperones. PMID : 23999302 : DOI : 10.1107/S090744491301500X Abstract >>
The Lon protease is ubiquitous in nature. Its proteolytic activity is associated with diverse cellular functions ranging from maintaining proteostasis under normal and stress conditions to regulating cell metabolism. Although Lon was originally identified as an ATP-dependent protease with fused AAA+ (ATPases associated with diverse cellular activities) and protease domains, analyses have recently identified LonC as a class of Lon-like proteases with no intrinsic ATPase activity. In contrast to the canonical ATP-dependent Lon present in eukaryotic organelles and prokaryotes, LonC contains an AAA-like domain that lacks the conserved ATPase motifs. Moreover, the LonC AAA-like domain is inserted with a large domain predicted to be largely �\-helical; intriguingly, this unique Lon-insertion domain (LID) was disordered in the recently determined full-length crystal structure of Meiothermus taiwanensis LonC (MtaLonC). Here, the crystal structure of the N-terminal AAA-like �\/�] subdomain of MtaLonC containing an intact LID, which forms a large �\-helical hairpin protruding from the AAA-like domain, is reported. The structure of the LID is remarkably similar to the tentacle-like prong of the periplasmic chaperone Skp. It is shown that the LID of LonC is involved both in Skp-like chaperone activity and in recognition of unfolded protein substrates. The structure allows the construction of a complete model of LonC with six helical hairpin extensions defining a basket-like structure atop the AAA ring and encircling the entry portal to the barrel-like degradation chamber of Lon.
|
5. |
Lin LH,
Chang CI,
Wu SH,
Liaw YC,
Wu WL,
Chang WH,
Yang CY,
Lin YC,
Lin YC,
Huang YY,
( 2012 ) A Lon-like protease with no ATP-powered unfolding activity. PMID : 22792246 : DOI : 10.1371/journal.pone.0040226 PMC : PMC3391209 Abstract >>
Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA(+) (ATPases associated with various cellular activities) module accommodated within a single polypeptide chain. They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA(+) motifs. Here we showed that these Lon-like proteases formed a clade distinct from LonA and LonB. Characterization of one such Lon-like protease from Meiothermus taiwanensis indicated that it formed a hexameric assembly with a hollow chamber similar to LonA/B. The enzyme was devoid of ATPase activity but retained an ability to bind symmetrically six nucleotides per hexamer; accordingly, structure-based alignment suggested possible existence of a non-functional AAA-like domain. The enzyme degraded unstructured or unfolded protein and peptide substrates, but not well-folded proteins, in ATP-independent manner. These results highlight a new type of Lon proteases that may be involved in breakdown of excessive damage or unfolded proteins during stress conditions without consumption of energy.
|