Crystal structure of scabies mite inactivated protease paralogue S-D1 (SMIPP-S-D1) (3H7T)

The scabies mite (Sarcoptes scabiei) is a parasitic mite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed Scabies Mite Inactivated Protease Paralogues (SMIPPs). The precise function of SMIPPs remains unclear. However, it has been suggested that these proteins may function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 and 2.0 A resolution respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical "canonical" fashion, and instead have evolved alternative functions in the lifecycle of the Scabies mite.

疥螨（Sarcoptes scabiei）是一种寄生螨，在全球范围内可对弱势社区与免疫功能受损患者造成严重发病负担。除该疾病引发的躯体不适外，疥螨感染还会通过皮肤破损部位促使链球菌类（Streptococcal）物种入侵，进而在受影响的社区中造成风湿热/心脏病的高流行率。疥螨可产生33种与尘螨第3组变应原（dust mite group 3 allergen）高度同源的蛋白质，这些蛋白隶属于S1样蛋白酶家族（S1-like protease family，类胰凝乳蛋白酶样（chymotrypsin-like））。然而，除其中一种外，其余所有这类蛋白的保守活性位点催化三联体（catalytic triad）均存在突变，据预测这些突变会使其丧失催化活性。这类分子因此被命名为疥螨灭活蛋白酶旁系同源物（Scabies Mite Inactivated Protease Paralogues，简称SMIPPs）。SMIPPs的确切功能目前仍不明确。不过有研究提出，这类蛋白可能通过结合并保护靶底物免受宿主免疫蛋白酶的切割来发挥功能，从而阻止宿主产生有效的免疫攻击。为了阐明SMIPP功能的结构基础，我们分别以1.85埃和2.0埃的分辨率解析了SMIPP-S-I1与SMIPP-S-D1的晶体结构。两种结构均呈现出典型的丝氨酸蛋白酶折叠（serine protease fold）模体，尽管在蛋白分子的大部分区域存在较大的结构变异。在两种结构中，催化三联体的突变以及保守的Tyr200残基对S1亚位点（S1 subsite）的遮蔽，据预测会阻止底物进入。据此我们证实，这两种蛋白酶均不具备催化活性。通过对催化残基及Tyr200进行定点诱变（site-directed mutagenesis）以恢复其功能的尝试均未成功。综合以上数据可知，SMIPPs已丧失以经典"规范"方式结合底物的能力，转而在疥螨的生命周期中演化出了其他功能。