Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function

Abstract The adipokine vaspin (serpinA12) is mainly expressed in white adipose tissue and exhibits various beneficial effects on obesity-related processes. Kallikrein 7 is the only known target protease of vaspin and is inhibited by the classical serpin inhibitory mechanism involving a cleavage of the reactive center loop between P1 (M378) and P1′ (E379). Here, we present the X-ray structure of vaspin, cleaved between M378 and E379. We provide a comprehensive analysis of differences between the uncleaved and cleaved forms in the shutter, breach, and hinge regions with relation to common molecular features underlying the serpin inhibitory mode. Furthermore, we point out differences towards other serpins and provide novel data underlining the remarkable stability of vaspin. We speculate that the previously reported FKGx1Wx2x3 motif in the breach region may play a decisive role in determining the reactive center loop configuration in the native vaspin state and might contribute to the high thermostability of vaspin. Thus, this structure may provide a basis for future mutational studies.

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Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement

International Journal of Molecular Sciences ◽

10.3390/ijms22179480 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9480

Author(s):

Jan Kotál ◽

Stéphanie G. I. Polderdijk ◽

Helena Langhansová ◽

Monika Ederová ◽

Larissa A. Martins ◽

...

Keyword(s):

Crystal Structure ◽

Ixodes Ricinus ◽

Blood Meal ◽

Coagulation Cascade ◽

Feeding Time ◽

Tick Saliva ◽

Reactive Center Loop ◽

Tick Borne Encephalitis ◽

Reactive Center

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P′ side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

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Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47036-6 ◽

1994 ◽

Vol 269 (44) ◽

pp. 27657-27662 ◽

Cited By ~ 2

Author(s):

D A Lawrence ◽

S T Olson ◽

S Palaniappan ◽

D Ginsburg

Keyword(s):

Reactive Center Loop ◽

Reactive Center ◽

Enzyme Recognition

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A reactive center loop–based prediction platform to enhance the design of therapeutic SERPINs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2108458118 ◽

2021 ◽

Vol 118 (45) ◽

pp. e2108458118

Author(s):

Wariya Sanrattana ◽

Thibaud Sefiane ◽

Simone Smits ◽

Nadine D. van Kleef ◽

Marcel H. Fens ◽

...

Keyword(s):

Cleavage Site ◽

Serine Proteases ◽

Protein C ◽

Activated Protein C ◽

Reactive Center Loop ◽

Physiological Processes ◽

Naturally Occurring ◽

Reactive Center ◽

Insight Into

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN–protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1’ residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence–function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4’ region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4’ region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4’ RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

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Truncated reactive center loop decrease the inhibitory activity of Antheraea pernyi serine protease inhibitor 6

Archives of Insect Biochemistry and Physiology ◽

10.1002/arch.21727 ◽

2020 ◽

Vol 105 (1) ◽

Author(s):

Guobao Wang ◽

Shuang Na ◽

Li Qin

Keyword(s):

Protease Inhibitor ◽

Serine Protease ◽

Inhibitory Activity ◽

Serine Protease Inhibitor ◽

Antheraea Pernyi ◽

Reactive Center Loop ◽

Reactive Center

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The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix

Journal of Biological Chemistry ◽

10.1074/jbc.m412043200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22356-22364 ◽

Cited By ~ 50

Author(s):

Ruby H. P. Law ◽

James A. Irving ◽

Ashley M. Buckle ◽

Katya Ruzyla ◽

Marguerite Buzza ◽

...

Keyword(s):

Crystal Structure ◽

High Resolution ◽

Tumor Suppressor ◽

Physiological Role ◽

Human Tumor ◽

Salt Bridge ◽

Structural Data ◽

Crystal Forms ◽

Reactive Center ◽

Α Helix

Maspin is a serpin that acts as a tumor suppressor in a range of human cancers, including tumors of the breast and lung. Maspin is crucial for development, because homozygous loss of the gene is lethal; however, the precise physiological role of the molecule is unclear. To gain insight into the function of human maspin, we have determined its crystal structure in two similar, but non-isomorphous crystal forms, to 2.1- and 2.8-Å resolution, respectively. The structure reveals that maspin adopts the native serpin fold in which the reactive center loop is expelled fully from the A β-sheet, makes minimal contacts with the core of the molecule, and exhibits a high degree of flexibility. A buried salt bridge unique to maspin orthologues causes an unusual bulge in the region around the D and E α-helices, an area of the molecule demonstrated in other serpins to be important for cofactor recognition. Strikingly, the structural data reveal that maspin is able to undergo conformational change in and around the G α-helix, switching between an open and a closed form. This change dictates the electrostatic character of a putative cofactor binding surface and highlights this region as a likely determinant of maspin function. The high resolution crystal structure of maspin provides a detailed molecular framework to elucidate the mechanism of function of this important tumor suppressor.

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