Báo cáo khoa học: Crystal structure of a subtilisin-like serine proteinase from a psychrotrophic Vibrio species reveals structural aspects of cold adaptation

Crystal Structure of a Subtilisin-like Serine Proteinase from a Psychrotrophic Vibrio Species Reveals Structural Aspects of Cold Adaptation

Tác giả: Jóhanna Arnórsdóttir, Magnús M. Kristjánsson, Ralf Ficner

Lĩnh vực: Sinh hóa, Cấu trúc protein

Nội dung tài liệu: Nghiên cứu này trình bày cấu trúc tinh thể của một enzyme serine proteinase thuộc nhóm subtilisin, được phân lập từ vi khuẩn Vibrio sp. PA-44, một sinh vật ưa lạnh. Đây là cấu trúc đầu tiên của một enzyme subtilisin thích nghi với môi trường lạnh được xác định. Việc phân tích cấu trúc này giúp làm sáng tỏ các nguyên tắc phân tử cơ bản cho sự thích nghi nhiệt độ của enzyme. Cấu trúc của Vibrio proteinase được so sánh với các enzyme tương đồng thuộc nhóm ưa ấm (mesophilic) và ưa nhiệt (thermophilic) để xác định các đặc điểm cấu trúc có thể đóng góp vào khả năng thích ứng với nhiệt độ thấp. Các yếu tố như bề mặt protein tiếp xúc với dung môi, sự sắp xếp của các gốc kỵ nước, mật độ tương tác ion và liên kết hydro, cũng như vai trò của các ion canxi, đều được xem xét. Nghiên cứu này cung cấp cái nhìn sâu sắc về cách các enzyme có thể duy trì chức năng và độ ổn định trong các điều kiện môi trường khắc nghiệt.

Mục lục chi tiết:

• Keywords
• Correspondence
• Database
• (Received 30 September 2004, revised 26 November 2004, accepted 9 December 2004)
• doi:10.1111/j.1742-4658.2005.04523.x
• The crystal structure of a subtilisin-like serine proteinase from the psychrotrophic marine bacterium, Vibrio sp. PA-44, was solved by means of molecular replacement and refined at 1.84 Å.
• This is the first structure of a cold-adapted subtilase to be determined and its elucidation facilitates examination of the molecular principles underlying temperature adaptation in enzymes.
• The cold-adapted Vibrio proteinase was compared with known three-dimensional structures of homologous enzymes of meso- and thermophilic origin, proteinase K and thermitase, to which it has high structural resemblance.
• The main structural features emerging as plausible determinants of temperature adaptation in the enzymes compared involve the character of their exposed and buried surfaces, which may be related to temperature-dependent variation in the physical properties of water.
• Thus, the hydrophobic effect is found to play a significant role in the structural stability of the meso- and thermophile enzymes, whereas the cold-adapted enzyme has more of its apolar surface exposed.
• In addition, the cold-adapted Vibrio proteinase is distinguished from the more stable enzymes by its strong anionic character arising from the high occurrence of uncompensated negatively charged residues at its surface.
• Interestingly, both the cold-adapted and thermophile proteinases differ from the mesophile enzyme in having more extensive hydrogen- and ion pair interactions in their structures; this supports suggestions of a dual role of electrostatic interactions in the adaptation of enzymes to both high and low temperatures.
• The Vibrio proteinase has three calcium ions associated with its structure, one of which is in a calcium-binding site not described in other subtilases.
• Microorganisms inhabit the most diverse environments on earth.
• Extremophiles are microorganisms that have adapted to environmental conditions regarded by humans as falling outside the normal range in terms of temperature, pressure, salinity or pH.
• Extremophiles have had to develop strategies to deal with environmental stress, mainly by molecular adaptation of their cell inventory.
• Of major importance in adapting to extreme environmental conditions is the optimization of protein function and stability.
• Enzymes from extremophiles are essentially like their mesophilic counterparts, sharing the same overall fold and catalysing identical reactions via the same mechanisms, while having adopted different traits regarding kinetic and structural properties.
• Therefore, they provide excellent tools for examining the molecular basis of different protein properties, as well as the relation between structure and function in enzymes.
• Regarding temperature, organisms have been isolated from places with temperatures as high as 113 °C [1] and biological activity has been detected in microbial samples at temperatures as low as -20 °C [2].
• Thermo- and hyperthermophiles face the challenge of keeping their macromolecules functional under the environmental stress imposed by extreme thermal motion.
• As a response, they have evolved enzymes that are highly stable against heat and other denaturants.
• The increased stability of enzymes from thermo- and hyperthermophiles is considered to reflect structural rigidity, which in turn would account for their poor catalytic efficiency at low temperatures.
• The properties of thermophilic enzymes have aroused great interest as they have potential in biotechnology and diverse industrial processes [3,4].
• In addition, the production of thermophilic recombinant enzymes is facilitated by their relatively straightforward overexpression and purification, which makes them feasible candidates for various biochemical experiments as well as for crystal structure determination.
• These factors have enhanced research on thermostability, which has been studied extensively in the past, mainly by comparing the structural properties of thermo- and mesophilic enzymes, as well as by using mutagenic experiments [5].
• In contrast to enzymes from thermophiles, cold-adapted enzymes are relatively poorly examined, in particular considering their extensive distribution and occurrence in our biosphere.
• Organisms occupying permanently cold areas that dominate the earth’s surface, collectively called psychrophiles, have to rely on enzymes that can compensate for low reaction rates at their physiological temperatures.
• The properties that characterize and distinguish cold-adapted enzymes from enzymes originating at higher temperatures are their increased turnover rate (kcat) and inherent higher catalytic efficiency (kcat/Km) at low temperatures [6].
• It is assumed that optimization of the catalytic parameters in cold-adapted enzymes is accomplished by developing increased structural flexibility, allowing the conformational changes required for catalysis at low temperatures [7].
• In recent years, a few crystal structures of cold-adapted enzymes have been determined [8-16].
• These structures have served as a basis in comparative studies on structural aspects of cold adaptation.
• Also, information from site-directed mutagenesis experiments, homology modelling and directed evolution has been used in an effort to shed light on the molecular principles underlying the adaptation of enzymes to low temperatures [17-24].
• In general, regardless of whether research is directed at thermo- or psychrophilic adaptation, the results show that each protein family adopts its own strategies for coping at extreme temperatures.
• Although no general rules have been found to apply in temperature adaptation in enzymes, some structural tendencies have emerged.
• The most frequently reported features related to temperature adaptation, going from higher to lower temperatures, are a reduced number of noncovalent intra- and intermolecular interactions, less compact packing of the hydrophobic core, an increased apolar surface area, decreased metal ion affinity, longer surface loops and a reduced number of prolines in loops [5,6,8,25-28].
• In general, in naturally occurring enzymes, a correlation is seen between catalytic efficiency at low temperatures and susceptibility to heat and other denaturants [29].
• However, using directed evolution methods, mutants have been obtained with changes in one of the properties, stability or catalytic efficiency, indicating that these properties are not essentially interlinked [22,23].
• The observed instability of cold-adapted enzymes is regarded not as a selected for property, but rather as a consequence of the reduction in stabilizing features arising from the need for increased flexibility to maintain catalytic efficiency at low temperatures [30].
• Structural flexibility in cold-adapted enzymes is, as yet, a poorly defined term for which little direct experimental evidence is available.
• Attempts to assess and compare the structural flexibility of a psychrophilic α-amylase and more thermostable homologues using dynamic fluorescence quenching supported the idea of an inverse correlation between protein stability and structural flexibility [31].
• Comparisons of hydrogen-deuterium exchange rates as a way of estimating flexibility in enzymes originating at different temperatures [32] have supported the idea of ‘corresponding states’ [33], which assumes that, at their physiological temperatures, enzymes possess comparable flexibility and a structural stability adequate to maintain their active conformation.
• In order to improve the understanding of the structural principles of temperature adaptation we studied a subtilisin-like serine proteinase from the psychrotrophic marine bacterium, Vibrio sp. PA-44.
• The Vibrio proteinase belongs to the proteinase K family and has a high sequence identity of 60-87% with several meso- and thermophilic family members [34].
• Furthermore, it has 41% sequence identity and 57% similarity with proteinase K, the best characterized representative of this protein family, the three-dimensional structure of which has been determined to atomic resolution [35].
• The Vibrio proteinase has been identified as showing clear cold-adaptive traits in comparison with its meso- and thermophilic homologues [36].
• Thorough sequence and computer model comparisons performed on the Vibrio proteinase and its most closely related meso- and thermophilic enzymes have revealed some differences, possibly relevant to temperature adaptation [34].
• The results have given rise to ongoing mutagenic research in which single and combined amino acid substitutions aimed at increasing the stability of the Vibrio proteinase are being tested.
• Elucidation of the Vibrio proteinase structure, the first structure of a cold-adapted subtilis e to be determined, enables a more focused examination of plausible determinants of different temperature adaptation among subtilases.
• We crystallized the cold-adapted Vibrio proteinase in the presence of bound inhibitor, phenyl-methyl-sulfonate, and the structure was refined at 1.84 Å resolution.
• In order to identify parameters that might be important with respect to cold adaptation we analysed and compared structural features in Vibrio proteinase and the two most closely related enzymes of known three-dimensional structure, proteinase K from the mesophilic fungi Tritirachium album Limber and thermitase from the thermophilic eubacterium Thermoactinomycet es vulgaris.
• Results
• The crystal structure of the Vibrio proteinase
• The obtained Vibrio proteinase crystals formed clusters of needles, which transformed into thin platelets within a few days.
• The crystals belong to space group P2₁ with unit cell dimensions_of a = 43.2 Å, b = 36.9 Å, c = 140.5 Å and ẞ= 97.8°.
• The Matthews coefficient [37] (Vm = 1.9 ų/Da) suggested two molecules in the asymmetric unit with a solvent content of 36.3%.
• The structure was determined by molecular replacement using a homology model based on the known structure of proteinase K (PDB accession number, 1IC6) as a search model.
• The crystallized 30 kDa catalytic domain of Vibrio proteinase encompasses amino acids 140-420 of the 530 amino acid prepro-enzyme [34].
• The model was refined at a resolution of 1.84 Å with an R-factor of 14.1% and an Rfree value of 19.6% (Table 1).
• Figure 1 shows the three-dimensional structure of Vibrio proteinase, hereafter referred to as 1SH7 according to its PDB accession number.
• The structure shows the α/β scaffold characteristic of subtilisin-like serine proteinases.
• It consists of six α helices, one 3/10 helix, a βsheet made of seven parallel strands and two βsheets made of two antiparallel strands (Fig. 1B).
• Determination of the structure confirms the presence of three previously predicted disulfide bonds, Cys67-Cys99, Cys163-Cys194 and Cys277-Cys281 [34].
• Three calcium-binding sites are found in 1SH7, two of which were predicted based on sequence alignments and one as yet not described in other subtilases.
• The active site of 1SH7 consists of the catalytic triad Asp37, His70 and Ser220, and substrate recognition and binding sites that are well conserved among subtilases [38].
• The substrate-binding site in 1SH7 appears on the surface as a relatively distinct cleft (see below, Table 1. Data collection and refinement statistics for 1SH7. Numbers in parenthesis refer to the highest resolution shell.
• Space group
• Unit cell parameters
• Number of reflections
• Unique reflections
• Completeness (%)
• Rsyma (%)
• Average Ι/σ
• Refinement statistics
• Resolution range (Å)
• Rcryst/Rfreeb (%)
• Rms deviation from ideality
• Bonds (Å)/angles (°)
• Average B-values (Ų)
• Protein/water/PMSF/Ca2+
• Ramachandran plot
• Most favoured, additional, generously allowed (%)
• ‘Surface properties and packing’) in which the substrate is accommodated by forming a triple-stranded antiparallel βsheet with residues of the S4- and S3-binding sites (nomenclature of subsites, S4-S2′, is according to Schechter and Berger [39]).
• The bottom of the S1 substrate-binding pocket is made up of residues A154-A155-G156 and the oxyanion hole residue N157.
• The substrate-binding cleft appears to be relatively open with T105 at the rim of S4; in many subtilases this site is occupied by a larger residue, typically a tyrosine (e.g. subtilisin and proteinase K), which is assumed to form a flexible lid on the S4 pocket [40].
• Overall structure comparison with related enzymes from meso- and thermophiles
• A 0.98 Å resolution structure of proteinase K (PDB accession number 1IC6) and a 1.37 Å resolution structure of thermitase (PDB accession number 1THM), were used for structural comparison with 1SH7.
• The high resolution of all three structures allows reasonable comparison with respect to the quality of the models.
• Pairwise least square superposition of the three structures, with a cut-off distance of 3.5 Å showed that 85-93% of the Ca-atoms lie at common positions and gave a root mean square deviation of 0.84–1.21 Å (Table 2, Fig. 2).
• The structural resemblance with Table 2. Pairwise superposition of Ca-atoms in 1SH7, 1IC6 and 1THM with a cut-off of 3.5 Å.
• Number of residues
• Aligned residues
• Identities
• Root mean square deviation (Å)
• regard to root mean square deviation, fraction of common Ca-atoms and the amino acid sequence identity, is in the order 1SH7-1IC6 > 1SH7-1THM > 1IC6-1THM.
• The distance deviations of the superposed structures and the locations of insertions and/or deletions are restricted to a few parts of the structure.
• The most distinct differences are seen in the N- and C-terminal regions, where 1THM aligns poorly with both 1SH7 and 1IC6.
• The C-termini of 1IC6 and 1SH7 also diverge; the last four residues of 1IC6 are not equivalent to residues in 1SH7.
• Furthermore, 1SH7 has an extended C-terminus relative to 1IC6.
• The four regions that deviate considerably owing to multiple residue insertions and deletions are marked in Fig. 2 as described below.
• First, a surface loop region, Phe57-Asn68 in 1SH7 does not align with 1IC6.
• This loop is identical in 1SH7 and 1THM and hosts a calcium-binding site that has been described as a medium-strong calcium-binding site in thermitase [41].
• Second, relative to both 1THM and 1SH7, 1IC6 has an insertion in an extended surface loop, residues 119–125 in 1IC6.
• This surface loop in 1IC6 contains some plausible stabilizing features, a disulfide bridge, Cys34-Cys123, and a salt bridge, Asp117-Arg121.
• Third, a loop region connecting α helices E, carrying the Ser of the catalytic triad, and the succeeding α helix F is not well conserved among the enzymes and the structures are accordingly variable.
• Fourth, 1SH7 contains a new calcium-binding site.
• This part of the structure is noticeably different from the corresponding regions in proteinase K and thermitase.
• If the allowed distance between equivalent Ca-atoms is defined as being within 2 Å, the ratio of Ca-atoms common to 1SH7 and the other two structures remains > 80%.
• The high structural homology of these enzymes which originate at different temperatures gives an opportunity to examine structural features that might contribute to their different temperature adaptation.
• Charged residues and ion pairs
• Thermitase contains 30 charged side chains, whereas proteinase K and the Vibrio proteinase each contain 38.
• The Vibrio proteinase differs from the enzymes with which it is compared in that it has a higher proportion of negatively charged side chains (Table