Roudiak S G, Shrader T E
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 1998 Aug 11;37(32):11255-63. doi: 10.1021/bi980945h.
Lon protease homologues contain a poorly conserved N-terminal region of variable length. To better understand the role of the N-terminal region of Lon in the complicated reaction cycle of ATP-dependent protein degradation, we expressed and characterized mutants of the Lon protease from Mycobacterium smegmatis (Ms-Lon) lacking 90, 225, and 277 N-terminal residues (N-G91, N-E226, and N-I278, respectively). N-I278 displayed neither peptidase nor ATPase activity despite the fact that it was stable and soluble in vivo, had a near-wild-type CD spectrum, and the deleted residues included neither the catalytic nucleophile for peptide bond hydrolysis (S675) nor the ATP binding regions. N-G91 and N-E226 retained peptidase activities against small unstructured peptides that were stimulated, to near-wild-type levels, by the Ms-Lon substrate protein alpha-casein. By contrast, N-G91 and N-E226 retained basal ATPase activities, but these activities were only stimulated weakly by alpha-casein. Ms-Lon, N-E226, and N-G91 all exhibited low-level peptidase activity in assays containing nonhydrolyzed nucleotide analogues. However, these peptidase activities were stimulated strongly by alpha-casein in the case of Ms-Lon but weakly by alpha-casein in the cases of N-G91 and N-E226. Strikingly, despite the near-wild-type peptidase activities of N-G91 and N-E226, both were severely impaired in their degradation of the Ms-Lon protein substrates alpha-casein in vitro and RcsA in vivo. Overall, N-G91 and N-E226 displayed catalytic properties similar to Escherichia coli Lon (Ec-Lon) in the presence of the PinA inhibitor, suggesting that PinA inhibits Ec-Lon protease by inhibiting the function of Ec-Lon's N-terminal region. In vivo protease assays further revealed that, in contrast to the inactive Ms-Lon point mutant S675A, N-G91 and N-E226 did not reduce the cellular activity of RcsA. This same defect was observed previously for Ms-Lons with multiple mutations in their peptidase active sites. We conclude that proteolytically inactive mutants of Ms-Lon retain the ability to reduce the cellular activity of RcsA but that both the N-terminal region and the peptidase active site region of Ms-Lon are required for this activity of wild-type Ms-Lon. The inabilities of N-G91 and N-E226 to degrade larger protein substrates and to reduce the cellular activity of RcsA were not the result of drastic alterations in their quaternary structures. Gel filtration profiles of N-G91 and N-E226 revealed that each was primarily tetrameric, with an increased percentage of dimeric species and a decreased percentage of trimeric species relative to Ms-Lon. The observed shifts in the dimer/trimer ratios of the N-terminal truncation mutants suggest that the Ms-Lon tetramer contains two types of subunit-subunit interactions.
Lon蛋白酶同源物含有长度可变且保守性较差的N端区域。为了更好地理解Lon蛋白酶N端区域在ATP依赖性蛋白质降解复杂反应循环中的作用，我们表达并鉴定了耻垢分枝杆菌（Ms-Lon）Lon蛋白酶缺失90、225和277个N端残基的突变体（分别为N-G91、N-E226和N-I278）。尽管N-I278在体内稳定且可溶，具有近乎野生型的圆二色光谱，并且缺失的残基既不包括肽键水解的催化亲核试剂（S675）也不包括ATP结合区域，但它既没有肽酶活性也没有ATP酶活性。N-G91和N-E226保留了针对小的无结构肽的肽酶活性，这些活性被Ms-Lon底物蛋白α-酪蛋白刺激至近乎野生型水平。相比之下，N-G91和N-E226保留了基础ATP酶活性，但这些活性仅被α-酪蛋白微弱刺激。Ms-Lon、N-E226和N-G91在含有非水解核苷酸类似物的测定中均表现出低水平的肽酶活性。然而，在Ms-Lon的情况下，这些肽酶活性被α-酪蛋白强烈刺激，而在N-G91和N-E226的情况下，被α-酪蛋白微弱刺激。令人惊讶的是，尽管N-G91和N-E226具有近乎野生型的肽酶活性，但它们在体外降解Ms-Lon蛋白底物α-酪蛋白和在体内降解RcsA的能力均严重受损。总体而言，N-G91和N-E226在存在PinA抑制剂的情况下表现出与大肠杆菌Lon（Ec-Lon）相似的催化特性，这表明PinA通过抑制Ec-Lon N端区域的功能来抑制Ec-Lon蛋白酶。体内蛋白酶测定进一步表明，与无活性的Ms-Lon点突变体S675A不同，N-G91和N-E226并没有降低RcsA的细胞活性。之前在肽酶活性位点有多个突变的Ms-Lon中也观察到了同样的缺陷。我们得出结论，Ms-Lon的蛋白水解无活性突变体保留了降低RcsA细胞活性的能力，但Ms-Lon的N端区域和肽酶活性位点区域对于野生型Ms-Lon的这种活性都是必需的。N-G91和N-E226无法降解较大的蛋白质底物以及降低RcsA的细胞活性，并非其四级结构发生剧烈改变的结果。N-G91和N-E226的凝胶过滤图谱显示，相对于Ms-Lon，它们每个主要是四聚体，二聚体物种的百分比增加，三聚体物种的百分比降低。N端截短突变体二聚体/三聚体比例的观察到的变化表明，Ms-Lon四聚体包含两种类型的亚基-亚基相互作用。
