Amino acid composition of the mutant proteins produced in E. coli. A122Q. I119A/F120A ..... Daniel J. Strydom for amino acid analyses. Parts of this work were ... Shapiro, R., Riordan, J. F. & Vallee, B. L. (1986) Biochemistry 25,. 3527-3532. 4.
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3243-3247, April 1996
Biochemistry
The C-terminal region of human angiogenin has a dual role in
enzymatic activity
(angiogenesis/RNase/hydrophobic interactions/mutagenesis) NELLO RUSSO, VALENTINA NOBILE, ALBERTO DI DONATO*, JAMES F. RIORDAN,
AND
BERT L. VALLEEt
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115
Contributed by Bert L. Vallee, December 26, 1995
occupied by the side chains of Gln-117 and Glu-118, respectively. Kinetic results with hAng derivatives mutated at Gln-
The ribonucleolytic activity of angiogenin ABSTRACT (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123, which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.
117 demonstrate that the obstruction also exists in solution and
suggest that Ang undergoes a conformational change to allow
substrate binding and catalysis to occur (9). Because the enzymatic activity of Ang is essential to its angiogenicity, the conformational change may also be part of its biological mode of action: Ang may be a latent factor subject to allosteric activation in vivo. Ang's subsequent action on a specific
substrate at its target site could then initiate the process of endothelial cell proliferation and blood vessel formation. Any such conformational change would require both disruption of the intramolecular interactions that stabilize the inactive structure and establishment of new interactions. As a first step in the structural characterization of this change, additional sitedirected mutagenesis and kinetic analysis have been employed to examine the intramolecular interactions that stabilize the inactive conformation of hAng. Two hydrophobic residues, Ile-119 and Phe-120-located in the middle of the C-terminal helix-contribute substantially to this stabilization. Moreover, the last three residues of the polypeptide chain (Arg-121, Arg-122, and Pro-123), which do not appear to form contacts with the rest of the protein structure, seem to constitute an active center subsite that participates in binding peripheral components of polymeric substrates.
Angiogenin (Ang) is a blood vessel-inducing protein (1, 2) unique among angiogenic factors owing to its exhibition of ribonucleolytic activity (3). Originally isolated from a medium conditioned by human colon adenocarcinoma cells (1), Ang was subsequently found to be expressed by a variety of tumor cell lines as well as by normal cells (4, 5), suggesting that in addition to tumor angiogenesis, it might have other functions. The ribonucleolytic activity of Ang was first hypothesized based on its sequence similarity to bovine pancreatic RNase A (RNase A) (6) and was detected only through the use of highly sensitive enzymatic assays (3). Like RNase A, Ang catalyzes the endonucleolytic cleavage of RNA and oligoribonucleotides on the 3' side of pyrimidines (7) via a transphosphorylation/ hydrolysis mechanism, but with 104- to 106-fold lower efficiency (3, 8, 9). Despite this low level of ribonucleolytic activity, it is essential for the angiogenicity of Ang; all mutations that substantially decrease this enzymatic activity also markedly diminish the capacity of Ang to induce blood vessel formation (10-12). This strict correlation suggests that Anginduced neovascularization results from the catalytic action of Ang on an RNA molecule yet to be identified. The crystal structures of human Ang (hAng) (13) and bovine Ang (14) show an overall fold similar to that of RNase A (15), but they also reveal a striking difference that accounts in part for the low ribonucleolytic activity of the angiogenins. In the human and bovine proteins, the site that is spatially analogous to the pyrimidine binding pocket of RNase A is blocked and
MATERIALS AND METHODS Materials and General Procedures. hAng (
p CpA poly(C)* 0.49 ± 0.04 1 12.2 ± 0.2 0.7 ± 0.1 4.0 ± 0.2 1 12.2 ± 0.1 1.83 ± 0.03 4 3.6 40.4 ± 0.8 2.27 ± 0.07 3.0 + 0.1 0.53 ± 0.05 0.1 9.0 ± 0.2 0.35 ± 0.01 0.1 Assay conditions were as described in the legend of Table 2. Conditions for poly(C) were the same as for tRNA; those for cytidine 2'3'-cyclic phosphate (C>p) were the same as those for dinucleotides. Values of hAng kcat/Km for CpA, UpA, and CpG are from a previous study (25). Data are the mean ± SD. *Activities relative to hAng.
Enzyme hAng I119A/F120A Ang Des(121-123) Ang
Role of the Arg-121-Pro-123 Segment of hAng. The last three residues of the hAng polypeptide chain, Arg-121, Arg122, and Pro-123, do not appear to interact with the rest of the protein structure. These residues are not conserved in Angs from other species (24), but were studied nevertheless because of their proximity to the active site region. A truncated hAng derivative, devoid of the Arg-121-Pro-123 segment, was prepared and tested with various RNA substrates. Its activity toward dinucleotides and cytidine 2',3' cyclic phosphate is very similar to that of the intact protein (Table 3). When tested with tRNA and poly(C), however, it was 10-fold lower (Table 3). Because tRNA and poly(C) are polymeric substrates, a likely interpretation of these data is that the Arg-121-Pro-123 region constitutes a peripheral substrate binding subsite that is occupied and contributes to activity only with polymeric substrates.
In the crystal structure of hAng, the
Arg-121-Pro-123
segment is located away from the catalytic histidines, on the back of the B1 site, where it could possibly constitute an additional phosphate (Po) or base (Bo) binding site. A recent combined kinetic and modeling study of the ribonucleolytic center subsites of hAng did not reveal any involvement of the Arg-121-Pro-123 segment in either a Po or a Bo site (25). Kinetic data indicated that hAng lacks an effective Po site, and a model of the interactions between hAng and a polymeric substrate analogue pointed to Asp-41 and Lys-82 as the only candidates for a possible Bo site. It should be noted that this modeling study was based on the inactive conformation of hAng and did not consider any structural rearrangement other than the repositioning of Gln-117. The present study shows
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