Dissecting the role of interprotomer cooperativity in the activation of oligomeric high-temperature requirement A2 protein

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy–based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding–incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson’s disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.

Download Full-text

The crystal structure of Mycobacterium tuberculosis high-temperature requirement A protein reveals an autoregulatory mechanism

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18016217 ◽

2018 ◽

Vol 74 (12) ◽

pp. 803-809

Author(s):

Arvind Kumar Gupta ◽

Debashree Behera ◽

Balasubramanian Gopal

Keyword(s):

Crystal Structure ◽

Mycobacterium Tuberculosis ◽

High Temperature ◽

Catalytic Domain ◽

Pdz Domain ◽

Regulatory Pathways ◽

Inactive Conformation ◽

Temperature Requirement ◽

Autoregulatory Mechanism

The crystal structure of Mycobacterium tuberculosis high-temperature requirement A (HtrA) protein was determined at 1.83 Å resolution. This membrane-associated protease is essential for the survival of M. tuberculosis. The crystal structure reveals that interactions between the PDZ domain and the catalytic domain in HtrA lead to an inactive conformation. This finding is consistent with its proposed role as a regulatory protease that is conditionally activated upon appropriate environmental triggers. The structure provides a basis for directed studies to evaluate the role of this essential protein and the regulatory pathways that are influenced by this protease.

Download Full-text

The crystal structure of an essential high-temperature requirement protein HtrA1 (Rv1223) from Mycobacterium tuberculosis reveals its unique features

Acta Crystallographica Section D Structural Biology ◽

10.1107/s205979831800952x ◽

2018 ◽

Vol 74 (9) ◽

pp. 906-921 ◽

Cited By ~ 1

Author(s):

Khundrakpam Herojit Singh ◽

Savita Yadav ◽

Deepak Kumar ◽

Bichitra Kumar Biswal

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Mycobacterium Tuberculosis ◽

High Temperature ◽

Protein Quality ◽

Pdz Domain ◽

Survival Strategies ◽

Dimensional Structure ◽

Protease Domain ◽

Temperature Requirement

High-temperature requirement A (HtrA) proteins, which are members of the heat-shock-induced serine protease family, are involved in extracytoplasmic protein quality control and bacterial survival strategies under stress conditions, and are associated with the virulence of several pathogens; they are therefore major drug targets. Mycobacterium tuberculosis possesses three putative HtrAs: HtrA1 (Rv1223), HtrA2 (Rv0983) and HtrA3 (Rv0125). Each has a cytoplasmic region, a transmembrane helix and a periplasmic region. Here, the crystal structure of the periplasmic region consisting of a protease domain (PD) and a PDZ domain from an M. tuberculosis HtrA1 mutant (mHtrA1S387A) is reported at 2.7 Å resolution. Although the mHtrA1S387A PD shows structural features similar to those of other HtrAs, its loops, particularly L3 and LA, display different conformations. Loop L3 communicates between the PDs of the trimer and the PDZ domains and undergoes a transition from an active to an inactive conformation, as reported for an equivalent HtrA (DegS). Loop LA, which is responsible for higher oligomer formation owing to its length (50 amino acids) in DegP, is very short in mHtrA1S387A (five amino acids), as in mHtrA2 (also five amino acids), and therefore lacks essential interactions for the formation of higher oligomers. Notably, a well ordered loop known as the insertion clamp in the PDZ domain interacts with the protease domain of the adjacent molecule, which possibly aids in the stabilization of a trimeric functional unit of this enzyme. The three-dimensional structure of mHtrA1S387A presented here will be useful in the design of enzyme-specific antituberculosis inhibitors.

Download Full-text

Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2

Biochemical Journal ◽

10.1042/bj20021569 ◽

2003 ◽

Vol 371 (1) ◽

pp. 39-48 ◽

Cited By ~ 79

Author(s):

Gui-Ying NIE ◽

Anne HAMPTON ◽

Ying LI ◽

Jock K. FINDLAY ◽

Lois A. SALAMONSEN

Keyword(s):

Alternative Splicing ◽

High Temperature ◽

Short Form ◽

Genomic Structure ◽

Expression Patterns ◽

Pdz Domain ◽

Northern Blotting ◽

Specific Function ◽

Temperature Requirement ◽

Long Form

In the present study, we identified an additional member of the human high-temperature requirement factor A (HtrA) protein family, called pregnancy-related serine protease or HtrA3, which was most highly expressed in the heart and placenta. We cloned the full-length sequences of two forms (long and short) of human HtrA3 mRNA, located the gene on chromosome 4p16.1, determined its genomic structure and revealed how the two mRNA variants are produced through alternative splicing. The alternative splicing was also verified by Northern blotting. Four distinct domains were found for the long form HtrA3 protein: (i) an insulin/insulin-like growth factor binding domain, (ii) a Kazal-type S protease-inhibitor domain, (iii) a trypsin protease domain and (iv) a PDZ domain. The short form is identical to the long form except it lacks the PDZ domain. Comparison of all members of human HtrA proteins, including their isoforms, suggests that both isoforms of HtrA3 represent active serine proteases, that they may have different substrate specificities and that HtrA3 may have similar functions to HtrA1. All three HtrA family members showed very different mRNA-expression patterns in 76 human tissues, indicating a specific function for each. Interestingly, both HtrA1 and HtrA3 are highly expressed in the placenta. Identification of the tissue-specific function of each HtrA family member is clearly of importance.

Download Full-text

A distinct concerted mechanism of structural dynamism defines activity of human serine protease HtrA3

Biochemical Journal ◽

10.1042/bcj20190706 ◽

2020 ◽

Vol 477 (2) ◽

pp. 407-429 ◽

Cited By ~ 1

Author(s):

Saujanya Acharya ◽

Shubhankar Dutta ◽

Kakoli Bose

Keyword(s):

High Temperature ◽

Serine Protease ◽

Pdz Domain ◽

Cellular Functions ◽

Potential Therapeutic Target ◽

Concerted Mechanism ◽

Effector Molecules ◽

N Terminus ◽

Opening Up

Human HtrA3 (high-temperature requirement protease A3) is a trimeric multitasking propapoptotic serine protease associated with critical cellular functions and pathogenicity. Implicated in diseases including cancer and pre-eclampsia, its role as a tumor suppressor and potential therapeutic target cannot be ignored. Therefore, elucidating its mode of activation and regulatory switch becomes indispensable towards modulating its functions with desired effects for disease intervention. Using computational, biochemical and biophysical tools, we delineated the role of all domains, their combinations and the critical phenylalanine residues in regulating HtrA3 activity, oligomerization and specificity. Our findings underline the crucial roles of the N-terminus as well as the PDZ domain in oligomerization and formation of a catalytically competent enzyme, thus providing new insights into its structure–function coordination. Our study also reports an intricate ligand-induced allosteric switch, which redefines the existing hypothesis of HtrA3 activation besides opening up avenues for modulating protease activity favorably through suitable effector molecules.

Download Full-text

High temperature requirement A1 in cancer: biomarker and therapeutic target

Cancer Cell International ◽

10.1186/s12935-021-02203-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Mingming Chen ◽

Shilei Yang ◽

Yu Wu ◽

Zirui Zhao ◽

Xiaohan Zhai ◽

...

Keyword(s):

High Temperature ◽

Therapeutic Target ◽

Public Health Problem ◽

Diagnostic Biomarkers ◽

Temperature Requirement ◽

Incidence And Mortality ◽

A Cell ◽

Cell Type Specific ◽

Cancer Types

AbstractAs the life expectancy of the population increases worldwide, cancer is becoming a substantial public health problem. Considering its recurrence and mortality rates, most cancer cases are difficult to cure. In recent decades, a large number of studies have been carried out on different cancer types; unfortunately, tumor incidence and mortality have not been effectively improved. At present, early diagnostic biomarkers and accurate therapeutic strategies for cancer are lacking. High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease that is also a chymotrypsin-like protease family member originally discovered in bacteria and later discovered in mammalian systems. HtrA1 gene expression is decreased in diverse cancers, and it may play a role as a tumor suppressor for promoting the death of tumor cells. This work aimed to examine the role of HtrA1 as a cell type-specific diagnostic biomarker or as an internal and external regulatory factor of diverse cancers. The findings of this study will facilitate the development of HtrA1 as a therapeutic target.

Download Full-text

Role of temperature in regulating the timing of germination in Portulaca oleracea

Canadian Journal of Botany ◽

10.1139/b88-081 ◽

1988 ◽

Vol 66 (3) ◽

pp. 563-567 ◽

Cited By ~ 10

Author(s):

Jerry M. Baskin ◽

Carol C. Baskin

Keyword(s):

High Temperature ◽

Minimum Temperature ◽

Early Summer ◽

Portulaca Oleracea ◽

Late Spring ◽

Daily Maximum ◽

Temperature Requirement

The peak of germination of autumn-sown seeds of Portulaca oleracea was between 21 May and 21 June, when mean daily maximum and minimum temperatures were 30.7 and 17.4 °C, respectively. Fresh seeds collected in October germinated to 13 and 94% at 30:15 and 35:20 °C thermoperiods, respectively, in light, and to 0% in darkness. Seeds were buried in October 1975 and exhumed in December 1975 through September 1976. In light, exhumed seeds germinated to 69–100% at 30:15 and 35:20 °C,to 1–80% at 20:10 °C, and to 0–52% at 15:6 °C; in darkness they germinated to 5–55% at 30:15 and 35:20 °C and to0% at 20:10 and 15:6 °C. Germination at 20:10 °C did not exceed 50% until mid-April, and it did not exceed 50% at 15:6 °C until June. Fresh seeds were buried at 5, 15:6, 20:10, 25:15, 30:15, and 35:20 °C, and after 0, 1, 3, and 5 months, seeds from each temperature were tested in light and darkness at the five thermoperiods. The minimum temperature at which 50% or more of the seeds germinated in light decreased with an increase in afterripening temperature. The high temperature requirement for complete afterripening and for germination of partially afterripened seeds prevents germination of this summer annual in temperate regions until late spring and early summer.

Download Full-text