Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins -- Sitar et al. 103 (35): 13028 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Sitar et al. 10.1073/pnas.0605652103.

Supporting Information

Files in this Data Supplement:

Supporting Materials and Methods
Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Table 2
Supporting Table 3

Supporting Figure 5

Fig. 5. Sequence and structure alignment of human IGFBP1–6. The N and C domains studied are marked by light blue. Conserved residues are indicated by gray shading, and cysteines are in yellow. N-domains: Residues shown in white have no amino acid and structural homology between N-domains of IGFBP4 and -5. Residues that interact with IGF1 are highlighted in red (with primary sites underlined) and hydrophobic residues of the "thumb" segments are boxed in green. For L-domains: In magenta, protease cleavage sites reported until now, amino acids labeled in blue show the calpain-cleavage site (unpublished data). Cleavage occurs after the marked amino acid. C domains: residues that interact with IGF1 or NBP4 are underlined with residues that interact with the thumb in bold.

Supporting Figure 6

Fig. 6. Surface representations of the CBP4 (Left) and CBP1 structures (Right).

Supporting Figure 7

Fig. 7. Structure of the NBP4(1–92)/IGF1 complex. Heavy atom (A) and ribbon (B) plots of the complex; NBP4 is shown in blue, IGF1 in green. Residues shown in violet constitute the binding site for interaction with NBP4. Residues marked in red are determinants for binding to IGF-IR. The GCGCCXXC consensus motif is shown in magenta (B).

Supporting Figure 8

Fig. 8. Alignment of all available primary thumb sequences of IGFBPs from various animal species. Residues that are identical or conserved are shaded.

Table 3. Data collection and refinement statistics

Data collection	NBP4 (1–92)/IGF1	NBP4 (1–92)/IGF1/CBP1	NBP4(3–82)/IGF-I/CBP4
X-ray source	Rotating-anode	BW6, DESY, Hamburg, Germany	ID29, ESRF, Grenoble, France
Space group	P2₁	P2₁	C2
Cell constants, Å	a = 32.33	a = 71.28	a = 74.4
	b = 38.99 β = 99.89	b = 43.66 b= 91.67	b = 50.25 b? = 115.3
	c = 61.33	c = 81.15	c = 64.3
Resolution range, Å	30–2.5	20–2.8	30–2.1
Wavelength, Å	1.54	1.05	0.97
Observed reflections	6,728	91,185	83,038
Unique reflections	5,177	13,980	12,370
Whole range
Completeness, %	91.8	83.4	92.6
R _merge	5.2	10.0	3.3
I/ σ(I)	21.6	11.9	25.4
Last shell
Resolution range, Å	2.5–2.6	2.8–2.9	2.1–2.2
Completeness, %	87.2	60.1	68.8
R _merge	14.9	37	9.4
I/ σ(I)	6.8	4.5	8.4

Refinement
No. of reflections	5,086	12,605	12,315
R factor, %	22.0	25.3	19.9
R _free, %	27.0	34.7	25.6
Average B, Å²	40.5	51.8	20.5
Rms bond length, Å	0.01	0.03	0.007
Rms angles, °	1.37	3.18	1.08
Content of asymmetric unit
No. of protein complexes	1	2	1
No. of protein residues per atom	181/1,107	407/3,044	460/1,845
No. of solvent atoms	34	–	254

DESY, Deutsches Elektronen Synchrotron; ESRF, European Synchrotron Radiation Facility.

Supporting Materials and Methods

X-Ray Crystallographic Procedure.
The data for the N-terminal domain of insulin-like growth factor-binding protein 4 (NBP4)(1-92)/insulin-like growth factor (IGF)1 crystals were collected from shock-frozen crystals at a rotating-anode laboratory source. The structure was determined by molecular replacement using the Molrep program from the CCP4 suite (1). The structure of the complex of IGF1 and a fragment of the N-terminal domain of IGFBP4 (residues 3–82) (PDB entry 1WQJ) (2) was used as a probe structure. Rotation search in the Patterson space yielded one peak of height 12.11 s over the highest noise peak of 4.21 Å. Translation search gave a 14.47-s peak over the noise height of 4.49 s. The initial R factor of the model was 0.47. The model was completed and revised manually by using Xfit software (3). Arp/wArp was used to add solvent atoms (4). The structure was finally refined by the Refmac5 program. Final electron-density maps were of good quality; there were, however, no interpretable densities for residue Pro-63 and side chains of residues Glu-11, Glu-12, Lys-13, Arg-16, Thr-37, Leu-42, Glu-66, His-70, Gln-76, Met-80, Glu-81, and Leu-82 in the NPB4(1-92) model. The IGF1 model had no interpretable electron density for the region Gly-30–Pro-39 and side chains of Arg-50 and Glu-58. These parts were removed from the model. The final R crystallographic factor was 0.23 and R_free, 0.27.

The structure of the NBP4(1-92)/IGF1 complex was then used as a molecular replacement probe for the data of the NBP4(1-92)/IGF1/C-terminal domain of IGFBP (CBP)1(141-234) crystals. Rotation search in Patterson space yielded two peaks of heights 8.37 and 7.1 s over the highest noise peak of 4.3 s. Translation search gave a 6.67-s peak over the noise height of 3.82 s for the first complex and 10.92 s over 4.95 s for the second one. The initial R factor of the model was 0.47. Phases calculated at this point allowed building of a partial model of the missing CBP1 part; the structure was then refined by a subsequent use of Refmac5 and manual model building. Noncrystallographic symmetry was used to improve the process because the asymmetric unit contains two complexes. Because of the limited quality of the experimental data, it was not possible to refine the model of the whole complex below the R factor of 25.3, an R_free of 34.7, with acceptable stereochemistry. The regions Gln-166–Ile-173 and Asp-197–Gly-198 in chain G, Ala-163–Lys-175 and Glu-193–Ala-200 in chain H, Gly-30–Gln-40 in chain C, and Pro-28–Thr-41 in chain I have no interpretable electron density and were removed from the model as well as flexible side chains invisible on the electron-density map.

The refined model of NBP4/(1-92)/IGF1/CBP1, after removing part of the NBP4 residues, was then used for molecular replacement with the diffraction data of the NBP4/(3-82)/IGF1/CBP4(151-232) crystals. The data, although good quality up to 2.1 Å, did not allow building a structure using only the binary complex as a search model (10). The molecular replacement using NBP4/(1-92)/IGF1/CBP1 was, however, very clear. Rotation search in the Patterson space yielded a peak of height 8.59 s over the highest noise peak of 4.77 s. Translation search gave a 14.75-s peak over the noise height of 4.74 s. The initial R factor of the model was 0.48 and dropped rapidly to 0.43 after rigid body refinement. At this stage, the calculated phases were improved by the DM program, and the phases obtained were used for the automatic model building in Arp/wArp. Approximately 80% of the model was built automatically, further completed, and revised manually by using Xfit software. Arp/wArp was used to add solvent atoms. The final model encompasses residue Gly-151–His-229 of the C-terminal domain of IGFBP4, Ala-3–Leu-82 of the NBP4 and Pro-2–Leu-64 of IGF1. The region between Ser-34 and Ala-38 is missing in the model; IGF1 was cleaved in this region since the distance between Ser-34 and Ala-38 is >17 Å. This fact explains the difficulties in repeating the crystallization of the complex because this region is involved in many crystal contacts. Some solvent-exposed side chains are also missing in the model and were removed from the structure. The final R crystallographic factor was 0.20 and R_free, 0.26.

1. CCP4, Collaborative computational project, Number 4. (1994) Acta Crystallogr. D 50, 760-763.

2. Siwanowicz, I., Popowicz, G. M., Wisniewska, M., Huber, R., Kuenkele, K. P., Lang, K., Engh, R. A. & Holak, T. A. (2005) Structure (Cambridge, U.K.) 13, 155-167.

3. McRee, D. E. (1999) J. Struc. Biol. 125, 156-165.

4. Lamzin, V. S., Wilson, K. S. (1993) Acta Crystallogr. D 49, 129-149.