Structure of α-lytic protease complexed with its pro region

Sauter, Nicholas K.; Mau, Ted; Rader, Stephen D.; Agard, David A.

doi:10.1038/2919

Cited by 75 publications

(90 citation statements)

References 32 publications

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“…2B). The alpha-lytic protease also has a C-shape propeptide (23), but the interfaces between the propeptide and the catalytic domain for the two proteases are different. The concave surfaces of both the FTP and PepSY domains are lined by four antiparallel β-strands, whereas their convex centers consist of two α-helices packed against the above β-sheets to form the hydrophobic core.…”

Section: Resultsmentioning

confidence: 99%

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Gao

Wang²,

Yu³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new β-strand, nestling into two other β-strands. Simultaneously, the apparent T m of the autoprocessed complex increases 20°C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.zymogen conversion | zinc metalloproteases inhibition | intramolecular chaperone T he thermolysin (M4) family is comprised of a large number of zinc metalloproteases in the subclan MA(E), most of which have similar sequences and domain structures (1). The representative member of this family is thermolysin (EC 3.4.24.27), a 34.6-kDa thermostable metalloprotease secreted by Bacillus thermoproteolyticus (2). The amino acid sequence and the threedimensional structure of thermolysin were first determined in 1972 (3, 4). Since then, thermolysin and thermolysin-like proteases (TLPs) have been used as models in studying zinc metalloproteases (5, 6).TLPs are widely distributed in many species of microorganisms, and many TLPs are regarded as key pathogenesis factors that are responsible for some severe bacterial infections (7). For example, λ-toxin from Clostridium perfringens can degrade various human immune defense proteins (8). Vibriolysin from Vibrio vulnificus (9) and pseudolysin from Pseudomonas aeruginosa (10) can be lethally blood-poisonous to humans. Therefore, understanding the catalytic and maturation mechanisms of TLPs is very important for developing therapeutics to treat such infectious diseases.TLPs are synthesized as a precursor with a propeptide. TLPs are divided into two distinct groups according to the primary structure of their propeptides. Less than 30% of the total TLPs have short propeptides (∼50 residues), and more than 70% of TLPs have long propeptides (∼200 residues) (11, 12). The long propeptide in TLPs contains two distinguished regions of conservation: an FTP (fungalysin/thermolysin propeptide) domain and a PepSY [peptidase propeptide and YPEB (a protein encoded by Bacillus subtilis ypeB gene)] domain. To date, the PepSY domain has been found not only in TLPs, but also in many nonpeptidase proteins, wherein the presence of this domain may prevent detrimental protease ...

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Section: Resultsmentioning

confidence: 99%

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Gao

Wang²,

Yu³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…After the cleavage, the N terminus dissociates out of the active site and then forms a final conformation identical to the crystal structure. The idea was derived from the crystal structure of the precursor of the ␣-lytic protease, a serine protease, which underwent autocatalytic cleavage during maturation (37). This model implies that the Arg8 residue is important for the 3B/3C cleavage, but is not necessary for cleavage at the other junctions.…”

Section: Vol 76 2002 Active-site Residues In Chiba Virus 3c-like Prmentioning

confidence: 99%

Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease

Someya

Takeda

Miyamura

2002

J Virol

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The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.

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“…The structure of αLP bound by its pro region showed that the pro region is a two-domain C-shaped protein that binds to and surrounds the αLP C-terminal domain (27). Based on these results, it was hypothesized that much of the folding defect of αLP would be localized to the C-terminal domain.…”

mentioning

confidence: 99%

Functional modulation of a protein folding landscape via side-chain distortion

Kelch

Salimi

Agard

2012

Proc. Natl. Acad. Sci. U.S.A.

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Ultrahigh-resolution (<1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6°distortion along the side-chain, estimated to cost 4 kcal∕mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.protein distortion | protein structure | X-ray crystallography | protein stability | protein lifetime T he physicochemical forces that determine macromolecular structure and function are generally thought to be well understood. The predominant forces include well-studied hydrogen bonding, van der Waals, and ionic interactions. By contrast, bonded terms are largely ignored as it is commonly held that bond lengths and angles are at or very close to canonical values in the native state. This view is so widely adopted that nearly all crystallographic and NMR structure determination methods include restraints to optimal values. However, distortion of covalent bonds in protein sidechains away from their low energy configurations has now been noted in several ultrahigh-resolution structures (1, 2) (Fig. 1A), but no functional role for the distortion has been ascribed. We examine the functional consequences of side-chain distortion in α-lytic protease (αLP), in which a 0.83 Å structure (2) revealed a surprisingly large distortion of the aromatic ring of a conserved Phe (Phe228) by almost 6°from planarity ( Fig. 1B; about 2.6 standard deviations from the mean in Fig. 1A).The αLP sub-family of serine proteases is one of the best-studied examples of a kinetically stable protein (3, 4); i.e. a protein that...

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Structure of α-lytic protease complexed with its pro region

Cited by 75 publications

References 32 publications

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease

Functional modulation of a protein folding landscape via side-chain distortion

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